Anti aging agents and methods to identify them

ABSTRACT

The present invention discloses novel mechanisms in the aging process and describes novel methods for high-throughput screening to identify, detect, and purify agents to be used for improving mitochondrial function, maintaining the cell cycle-arrested state in senescent and post mitotic cells, and thus preventing or treating age-related diseases or disorders associated with accelerated mitochondrial function loss, telomere dysfunction, and/or deterioration of the growth-arrested state. The present invention also discloses a number of compounds or compositions identified from this method. The present invention further provides the use of low doses of rapamycin or its analogs as a mimic of caloric restriction in preventing age-related diseases or disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/168,311, filed on Apr. 10, 2009, and U.S.Provisional Application No. 61/168,335, filed on Apr. 10, 2009, both ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention is related to novel anti-aging agents, novel methods todetect or identify these agents, and use of the anti-aging agents soidentified for the prevention and/or treatment of age-related diseasesor disorders. The invention is also related to a novel method formeasuring the anti-aging biological concentration of anti-aging agentsin biological samples. In particular, the invention introduces the useof low doses of rapamycin or its analogs, among othertarget-of-rapamycin (TOR) inhibitors, as anti-aging agents mimickingcalorie restriction for preventing or treating various age-relateddiseases or disorders.

BACKGROUND OF THE INVENTION

Study of human aging processes is important, in part because manydiseases or conditions become more prominent among aged people, forexample, cancers, Alzheimer's disease, Parkinson's disease, stroke,heart failure, and heart attack, just to name a few, among which manystill lack effective preventive or treatment methods. Therefore, thesearch for more effective prevention or treatment methods for theage-related diseases or disorders through studies of animal agingprocesses has become one of the most important endeavors embarked by thescientific community in the last decade. Although abundant literaturehas contributed to the understanding of aging processes, fullunderstanding of the processes remains to be a major scientificchallenge faced by mankind. Given the increasing population of agedpeople around the world and the rising health care burden and costassociated therewith, systematic studies of the aging processes leadingto effective discovery of anti-aging agents for the prevention ortreatment of age-related diseases or disorders are becoming increasinglyimportant. This invention represents such a systematic approach aimingto provide effective methods for the discovery of anti-aging agents thatcan be developed into effective prevention and/or treatment ofage-related diseases or disorders.

Among various theories concerning the aging processes and the methodsderived from the theories that could be useful for the treatment ofage-related diseases, the nutrient signaling pathway (caloricrestriction), the mitochondria pathway (reactive oxygen species, orROS), and the telomere dysfunction theory are prominent.

Nutrient Signaling Pathway (Caloric Restriction) and Aging. Caloricrestriction (CR) has been recognized as the most practicable method toretard the rate of aging from yeast to mammals. CR has also been shownto reduce the incidence or delay the onset of age-related diseases suchas Parkinson's disease in a primate model (Maswood, N., et al., Proc.Natl. Acad. Sci. USA, 101:18171-6 (2004)), Alzheimer's disease (Qin, W.,et al., J. Alzheimer's Dis., 10:417-422 (2006)), hypertension and heartproblems in the Dahl-SS rat model (Seymour, E. M., et al., J. Mol. CellCardiol., 41:661-668 (2006)), fibrosis (Castello, L., et al., FASEB J.,19:1863-1865 (2005)), and kidney disease (Yu, B. P., et al., J.Gerontol., 37:130-141 (1982)). CR also inhibits a variety of spontaneousneoplasias and decreases the incident of human breast, colon andprostate cancers (reviewed in Platz, E. A., J. Nutr., 132:3471S-81S(2002); Steinbach, G., et al., Cancer Res., 54:1194-1197 (1994);Michels, K. B., et al., JAMA, 291:1226-30 (2004)).

The well-conserved kinase TOR (Target of rapamycin) integrates signalsfrom nutrients, mitogenic growth factors, energy, and stress to regulatecatabolic and anabolic processes (Fingar, D. C., et al., Oncogene,23:3151-3171 (2004)). In response to optimal growth factors andnutrients, mammalian TOR (mTOR) stimulates the cell's syntheticcapabilities (such as ribosome biogenesis and protein translationinitiation), leading to increases in cell mass and size and acceleratesproliferation (Kim, E., et al., Hum. Gene Ther., 14:1415-1428 (2003)).Conversely, inhibition of TOR by growth factor withdrawal, nutrientstarvation, or stress leads to the down-regulation of highenergy-consuming processes and inhibition of proliferation.

TOR pathway may play an important role in the life span extensioninduced by CR in budding yeast, Ceanorhabditis elegans and Drosophila(Kaeberlein, M., et al., Science, 310:1193-1196 (2005); Powers, R. W.,et al., Genes Dev., 20:174-84 (2006); Vellai, T., et al., Nature,426:620 (2003); Kapahi, P., et al., Curr. Biol., 14:885-890 (2004); Jia,K., et al., Development, 131:3897-3906 (2004)). As the function of TORis well conserved, its role in aging may also apply to humans.

The Mitochondrion/ROS and Aging. Mitochondria are cellular organellesresponsible for converting metabolic fuels (e.g., glucose and fattyacids) into a usable form of energy, adenosine 5′-triphosphate (ATP),through the process of oxidative phosphorylation. Mitochondria are alsoinvolved in other processes that are important for proper cellularfunction, including calcium homeostasis, intracellular signaltransduction, and the regulation of apoptosis.

The process of oxidative phosphorylation for ATP generation inmitochondria is also the main source of reactive oxygen species (ROS)within the cell (about 90% of total ROS in cells) (Balaban, R. S., etal., Cell, 120:483-495 (2005)). Under normal physiological conditions,ROS leaked during oxidative phosphorylation is estimated to represent1-5% of the oxygen consumed during this process (Chance, B., et al.,Physiol. Rev., 59:527-605 (1979)). Due to the limited repair capacity ofmitochondrial DNA (mtDNA) and the proximity to the oxidants,mitochondria are particularly vulnerable to accumulation of damages.Mutations in mtDNA then result in impaired function of oxidativephosphorylation, leading to increased ROS production and the subsequentaccumulation of more mutations. As ROS are highly reactive molecules andcan generate diverse damages in the cells, the ROS vicious cycle isbelieved to account for an exponential increase in oxidative damageduring aging, which results in a gradually functional decline thatcharacterizes the aging process.

ROS may be associated with many age-related diseases, for example,diabetes, cardiovascular disease, cancer and Parkinson's disease(Kovacic, P., et al., Curr. Med. Chem., 8:773-796 (2001); Aviram, M., etal., Am. J. Clin. Nutr., 71:1062-1076 (2000); Maassen, J. A., et al., J.Endocrinol. Invest., 25:477-484 (2002)). The fact that eukaryotesdevelop a host anti-oxidant defense system also supports the importantrole of endogenous ROS production (Mates, J. M., Toxicology, 153:83-104(2000)) and overexpression of superoxide dismutase and catalase extendslife span in Drosophila melanogaster (Orr, W. C., et al., Science,263:1128-1130 (1994)).

Previous studies indicate that mitochondrial integrity declines as afunction of age as monitored by decreases in mitochondrial membranepotential, mitochondrial number, and ATP generation/O₂ consumption(Hagen, T. M., et al., Proc. Natl. Acad. Sci. USA, 94:3064-3069, 1997;Greco, M., et al., FASEB J., 17:1706-1708 (2003)). Mutations inmitochondrial function cause a number of human genetic diseases withclinical manifestations including blindness, deafness, movementdisorders, dementias, cardiovascular disease, muscle weakness, renaldysfunction, and endocrine disorders. Furthermore, it has been reportedthat mice with a dramatic increase in mitochondrial DNA mutations (dueto a proof-reading-deficient mutation in mtDNA polymerase PolgA)exhibited a shorter life span, accompanied with certain premature agingphenotypes (Trifunovic, A., et al., Nature, 429:417-423 (2004)).Moreover, life span extension in yeast (Chronological life span) bydeletion of TOR1 and in C. Elegans by glucose restriction has beenreported to be via mitochondrial respiration (Bonawitz, N. D., et al.,Cell Metab., 5:233-235 (2007); Schulz, T. J., et al., Cell Metab.,6:280-293 (2007)). These results suggest the important role ofmitochondrial function in aging and age-related diseases in mammals.However, contradictory results have also been reported. For example,CR-induced life span extension in budding yeast was reported to beindependent of mitochondrial function (Kaeberlein, M., et al., PloSGenet., 1, e69 (2005)). Therefore, the role of mitochondria in the agingprocess remains to be unclear.

Telomeres, Senescence, Aging and Cancer. Telomeres are ends ofchromosomes consisting of G-rich repeated DNA sequences on one strand.The telomeres are bound by telomere binding proteins to protect themfrom being recognized as the naturally occurring double-stranded DNAbreaks (DSBs).

Dysfunctional telomeres can be caused by progressive shortening oftelomeres due to the internal problem of DNA replication by DNApolymerase and the lack of telomerase activity in most somatic cells inhumans. Eventually, the critically shortened telomeres cannot be boundby telomere proteins and are thus exposed as natural DSBs, whichactivate DNA damage responses and induce RB- and p53-dependent cellcycle arrest. This process is termed replicative senescence. Inaddition, senescence can be induced by oncogenes activation via the sameDNA damage responses, resulting in tumor suppression (Di Micco, R., etal., Nature, 444:638-642 (2006); Bartkova, J., et al., Nature,444:633-637 (2006)). Furthermore, DNA damage agents have also beenreported to trigger senescence as well.

Telomere dysfunction also occurs when there is a telomere bindingprotein defect. For example, expression of a dominant-negative TTAGGGrepeat binding factor 2 (TRF2), as well as knockdown of protection oftelomeres 1 (POT1), results in telomere dysfunction and DNA damagesignals (Karlseder, J., et al., Science, 283:1321-1325 (1999); Denchi,E. L., et al., Nature, 448:1068-1071 (2007); Guo, X., et al., EMBO J.,26:4709-4719 (2007)).

Long telomeres have been associated with longevity in humans, whileshort telomeres have been associated with cancers, idiopathic pulmonaryfibrosis and a variety of proliferative tissue disorders. For example,the telomerase mutation in human causes Dyskeratosis congenita andpatients typically die early of bone marrow failure.

Replication senescence has been shown to be a barrier for tumorprogression, since cancer cells require unlimited replication potential.Indeed, senescent markers are prominent in pre-malignant lesions butundetectable in advanced cancers in mouse models and in human cancers(Braig, M., et al., Nature, 436:660-665 (2005); Collado, M., et al.,Nature 436:642 (2005); Michaloglou, C., et al., Nature, 436:720-724(2005)). All cancers bypass senescence by activating telomerase oralternative telomere lengthening by recombination (Shay, J. W., et al.,Exp. Cell. Res., 209:45-52 (1993); Shay, J. W., et al., Eur. J. Cancer,33:787-791 (1997); Kim, N. W., et al., Science, 266:2011-2015 (1994);Bryan, T. M., et al., Nat. Med., 3:271-274 (1997)). The progression ofearly stage prostate cancer to malignance is blocked by senescence(Chen, Z., et al., Nature, 436:725-730 (2005)). Furthermore, thespontaneous tumorigenesis induced by telomere dysfunction in telomerasemutant mice Terc^(−/−) was shown to be inhibited by p53-mediatedsenescence (Cosme-Blanco, W., et al., EMBO Rep. 8:497-503 (2007)).Senescence is assumed to stop the cell cycle and facilitate repair, thusblocking further development of the initial lesions.

Senescence is also viewed as a major contributor to aging (Campisi, J.,Nat. Rev. Cancer., 3:339-49; Faragher, R. G., Biochem. Soc. Trans.,28:221-226 (2000)). For example, senescent cells increase with age inmammalian tissues (Campisi, J., Cell, 120:1-10 (2005)). Senescent cellshave been found at sites of age-related pathologies such asosteoarthritis and atherosclerosis (Price, J. S., et al., Aging Cell,1:57-65 (2002); Vasile E., et al., FASEB J., 15:458-466 (2001);Matthews, C., et al., Cir. Res., 99:156-164 (2006)). Moreover,chronically active p53 both promotes cellular senescence and acceleratesaging phenotypes in mice (Maier, B., et al., Genes Dev., 18: 306-319(2004); Tyner et al., Nature, 415:45-53 (2002)). Furthermore, it hasbeen shown that senescent cells secret proteins that facilitate tumorprogression and inflammation response (Coppe, J. P., et al.,PlosBiology, 6:2853-2868 (2008)). It has been proposed that theprogrammed senescence leads to age-related diseases and limited our lifespan (Blagosklonny, M. V., Cell Cycle, 5:2087-2102 (2006)). Therefore,anti-aging research from the telomere angle is currently focused onpreventing senescence.

Despite all the studies, the role of telomeres in aging process remainsunclear. For example, it cannot explain why a mouse has longer telomeresbut a shorter life span than a human. It is not clear if or howtelomeres work in the aging process of post-mitotic cells.

Other aging theories have also been proposed, for example, the proteindamage accumulation theory, the DNA mutation accumulation theory, andthe stem cell exhausting theory. Among the above, which theoriesrepresent the true nature of the aging processes and whether and/or howthey are related to each other are still unclear. Therefore, at least tosome degree, human aging processes remain to be a mystery.

Age-related diseases or disorders, such as cancers, cardiovasculardiseases, and neuronal degeneration diseases, are leading causes ofdeath in humans. Pharmaceutical agents for the treatment of theseage-related diseases or disorders are being searched according to thecurrent understanding of the specific diseases, due to the limitedunderstanding about the aging processes. As a result, to date, thesediseases have been studied independently of each other and disconnectedfrom the aging processes. Therefore, there is a need to develop asystemic approach based on the aging processes to the discovery of novelanti-aging agents for the prevention and treatment of age-relateddiseases or disorders.

SUMMARY OF THE INVENTION

The present invention provides the forgoing need by disclosing novelmechanisms in the aging processes and novel methods based the novelmechanisms to identify or detect anti-aging agents useful for theprevention and/or treatment of age-related diseases or disorders. Thenovel methods disclosed herein can be used to identify novel anti-agingagents rapidly by high-throughput screening using variouswell-understood yeast mutant models. In particular, the presentinvention describes the use of low doses of rapamycin or its analogs,among a number of other anti-aging agents identified by using themethods disclosed, for effective prevention and/or treatment of variousage-related diseases or disorders.

In one aspect, the present invention provides a method of identifying ordetecting an agent for preventing or treating an age-related disease ordisorder, the method comprising screening one or more compounds orcompositions against a senescence model system and monitoring theiranti-aging activity.

In another aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and measuring their activity against atleast one of components of the TOR/AMPK/Mitochondria/Senescence pathway.

In another aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the mitochondrial biogenesis pathway.

In another aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating aging or anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the AMPK pathway.

In another aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the senescence pathway, wherein the agentmaintains senescence or cell cycle-arrested state in post-mitotic cellsor prevents deterioration of mitochondria or cell death followingsenescence deterioration.

In another aspect, the present invention provides a method of preventingor treating an age-related disease or disorder, the method comprisingadministering to a subject in need thereof a composition comprising anagent identified according to any of the embodiments described in any ofother aspects of the present invention, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof.

In another aspect, the present invention provides a method of preventingor treating an age-related disease or disorder associated withdeterioration of telomeres and/or mitochondria, the method comprisingadministering to a subject in need thereof a composition comprising a5′-adenosine monophosphate-activated protein kinase (AMPK) activator, ora pharmaceutically acceptable salt, solvate, or prodrug thereof, whichdirectly or indirectly activates AMPK, increases mitochondrialbiogenesis, and maintains a cell cycle-arrested state in the senescentor post-mitotic cells of the subject.

In another aspect, the present invention provides a method of preventingor treating an age-related disease or disorder, the method comprisingadministering to a subject in need thereof a composition comprising atarget-of-rapamycin (TOR) inhibitor, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, wherein the TOR inhibitor (a) extendsreplication potential, (b) maintains senescence or cell cycle-arrestedstate in post-mitotic cells, or (c) prevents deterioration ofmitochondria or cell death following senescence deterioration. In apreferred embodiment of this aspect, said TOR inhibitor is a low dose ofrapamycin or an analog thereof.

In another aspect, the present invention provides a method for detectingan anti-aging agent in a biological sample, the method comprising usinga yeast senescence model.

In another aspect, the present invention provides a method fordetermining biological concentration of an anti-aging agent in abiological sample, the method comprising using a yeast senescence modeland a pre-established standard equation or curve of the anti-agingagent.

In another aspect, the present invention discloses that mitochondrialfunction plays an important role in maintaining senescence induced bytelomere dysfunction, and caloric restriction (CR) preventsdeterioration of the senescent state through the TOR/AMPK/mitochondrialpathway. This mechanism is conserved in both yeast and human models oftelomere dysfunction. The conserved mechanism thus allows the use oftelomere dysfunction models in yeast and in humans to search forpharmaceuticals that can promote mitochondrial function and thus preventor treat deterioration of senescence. Since many age-related diseasesare linked to mitochondrial dysfunction and/or telomere dysfunction,agents identified by this method can potentially be used to preventage-related diseases or disorders. Thus, in contrast to the currentlyprevalent anti-aging strategies, which are mainly focused on inhibitingsenescence by taking senescence as the major contributor to the agingprocess, the present invention introduces a novel strategy forpreventing or treating age-related diseases or disorders throughmaintaining senescence.

Other aspects and specific preferred embodiments of the presentinvention are described in more details in the following embodiments andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that limiting nutrient signaling inhibits cell deathinduced by cdc13-1p inactivation. (A) Glucose restriction and2-deoxy-glucose treatment inhibit cell loss induced by Cdc13pinactivation as assayed by colony formation assay. To induce telomeredysfunction, fresh overnight cultures of cdc13-1 were diluted into YEPDmedium with the indicated concentrations of glucose or 2-deoxyglucoseand incubated at 37° C. for 24 hrs. For colony formation assay, treatedcells were then serially diluted 10-fold in H₂O and 5 μL was spottedonto regular YEPD plates. The plates were incubated at 24° C. forsurvived cells to form colonies. The starting number of live-cells wasalso counted by this colony formation assay. (B) Nitrogen restrictioninhibits cell death induced by cdc13-1p inactivation as monitored by acolony formation assay. Cells were incubated in synthetic medium (SC) orSC-N (SC without amino acids and (NH₄)₂SO₄ as nitrogen sources) at 37°C. for 24 hrs. The number of survived cells was counted using colonyformation assay as described in 1A. (C-D) Inhibition of TOR by low dosesof rapamycin (labeled as Rapa) (below growth inhibition concentration)can prevent cell death induced by cdc13-1p inactivation. Cells wereincubated with the indicated concentrations of rapamycin in YEPD mediumat 37° C. for 24 hrs. The number of survived cells was measured by thecolony formation assay as in 1A. For growth curve, fresh overnightculture was diluted into YEPD medium and incubated at 24° C. in thepresence of the indicated concentrations of rapamycin. Cell Density(OD₅₉₅) was measured at the indicated time points and plotted againstthe time. (E) Deterioration of growth arrested cdc13-1 cells can bedelayed by low dose of rapamycin (1 nM) and glucose restriction (0.5%)as measured by counting surviving cells using the colony formationassay. The data represent an average of three experiments.

FIG. 2 shows that nutrient signaling does not interfere with the cellcycle arrest at G2/M, but maintains the G2/M-arrested state and preventscell death induced by inactivation of cdc13-1p. (A) Cells were incubatedat 37° C. to inactivated cdc13-1p, in YEPD medium, YEPD+1 nM rapamycinor YEPD with 0.5% glucose. At the indicated time points, an aliquot ofcells was taken out and fixed with 50% ethanol at −20° C. for about 4hrs, and then digested with 0.2 mg/mL RNase A in 50 mM Tris pH 7.6 at37° C. overnight. The cells were next washed with 50 mM Tris pH 7.6 andtreated with 40 μg/mL proteinase K at 55° C. for 2 hrs. After beingwashed again, cells were stained with 100 μg/mL propidium iodide for 20min in the dark prior to FACS (Fluorescence activated cell sorter)analysis. (B) Cell survival assay shows that the death of G2/M cellsinduced by inactivation of cdc13-1p for 2 hrs can still be prevented byrapamycin and glucose restriction. Cells were incubated at 37° C. for 2hours first, then diluted with a 37° C-YEPD medium, YEPD with rapamycinto make a final concentration of 1 nM, or with a 37° C-medium withoutglucose to make final 0.5% glucose. The cells were continuouslyincubated at 37° C. for 22 hrs. Surviving cells were counted using thecolony formation assay.

FIG. 3 shows that rapamycin (1 nM) and reduced glucose (0.5%) decreaseROS production by inactivation of cdc13-1p as measured bydihydrorhodamine 123 (Invitrogene) staining followed by FACS analysis(A) and decrease an apoptotic marker PS flipping as measured by annexinV-FITC binding followed by FACS analysis (B). Cells were treated underconditions as described in FIG. 1A and FIG. 1C. To measure ROS levels,the treated cells were incubated with 5 μg/mL dihydrorhodamine 123 inYEPD for 1 hr prior to FACS analysis. 10,000 cells were analyzed foreach sample. To measure PS flipping, the treated cells were resuspendedin a PBS buffer containing 1.1 M sorbitol and 2 mg/mL Zymolyase, andincubated at 37° C. for 20 min. The cells were then stained with annexinV-FITC and propidium iodide (PI) (BD Biosciences Pharmingen) in PBS(Phosphate Buffered Saline) containing 1.1 M sorbitol, followed by FACSanalysis. 10,000 cells were analyzed for each sample. Under theseconditions, the PI-negative population represents intact cells, thePI-negative-FITC-positive cells represent the apoptotic population, andthe PI-positive-FITC positive cells represent the late apoptotic ornecrosis population.

FIG. 4 shows that rapamycin and glucose restriction prevent cell deathinduced by inactivation of cdc13-1p through AMPK. (A) Deletion of theAMPK regulatory subunit Sip2p abolishes the preventive effect of glucoserestriction. (B) Deletion of the AMPK catalytic subunit Snf1p and theregulatory subunit Snf4p significantly decreases the preventive effectof rapamycin (1 nM). The cdc13-1sip2::Kan, cdc13-1snf1::Kan andcdc13-1snf4::Kan double mutant strains were generated by mating thesingle deletion mutant from the deletion library (from Invitrogen,Carlsbad, Calif.) with cdc13-1, followed by sporulating the diploid andselecting for temperature-sensitive and G418 (200 μg/mL)-resistantcolonies. Cells were treated as in FIG. 1A and FIG. 1C. Surviving cellswere monitored by the colony formation assay.

FIG. 5 shows that mitochondria play an important role in the preventiveeffect of nutrient limitation on cdc13-1 cell death. (A) Mitochondriadeficiency significantly inhibits the preventive effect of rapamycin andglucose restriction. Mitochondria deficient mutant ρ^(o) was generatedin cdc13-1 by growing cells in an ethidium containing YC medium to logphase for two days as described (Qi, H., et al, J. Biol. Chem., 278:15136-15141 (2003)). The cells were treated as indicated and asdescribed in FIG. 1A and FIG. 1C. Surviving cells were monitored bycolony formation assay. (B-C) Glucose restriction and rapamycintreatment increase mitochondrial mass. Fresh diluted overnight culturesin YEPD, YEPD+rapamycin (B) or YEPD, 0.5% glucose YEPD (C) wereincubated at 24° C. to for 4 hrs to log phase. Mitochondrial mass wasmeasured by staining the 60%-ethanol fixed cells using MitoTracker GreenFM, followed by FACS analysis.

FIG. 6 shows the mechanism of nutrient signaling through TOR, AMPK, andmitochondria in maintaining the cell cycle-arrested state and preventingcell death induced by telomere dysfunction in the cdc13-1 model.

FIG. 7 shows that the loss of the senescent WI-38 cells (human primaryfibroblast) is prevented by the treatment of 50 pM rapamycin, 250 μMAICAR, 20 μg/mL EGCG, 1.6 μg/mL GSE, reduced glucose (from 0.4% to0.2%), 20 μg/mL bilberry extract (BE), 1 μM AITC, and 12.5 μM2-deoxyglucose. AICAR and reduced glucose treatment were incubated withcells in a 2-day-on/8-day-off cycle, while the rest of agents were in a3-day-on/7-day-off cycle. The treatment started at passage 29. Themedium was refreshed every 3 days. After 56-days from passage 31(senescence), the cells were briefly fixed in 2% formaldehyde/0.2%glutaraldehyde and stained with 1 mg/mL 5-bromo-4-chloro-3-indolylβ-D-galactoside (X-gal) (in buffer containing 40 mM citric acid/sodiumphosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide, 150 mM NaCl, and 2 mM MgCl₂) at 37° C. for 18 hrs for thesenescence marker, cellular β-galactosidase activity (blue in colorpicture and dark grey in black/white picture). Surviving senescent cellswere viewed under a microscope.

FIG. 8 shows that low doses of rapamycin increase mitochondrial mass,improve mitochondrial membrane potential and decrease ROS levels inhuman fibroblasts. (A) WI-38 cells at passage 24 were treated withvarious doses of rapamycin in culture medium for 2 days. Formitochondrial mass measurements, the cells were fixed with 60% ethanolat −20° C., and then stained with MitoTracker Green FM (Invitrogene) for30 min prior to FACS analysis. (B) Human lymphoblastoid L40 cells weretreated with various doses of rapamycin for two days. For mitochondrialmembrane potential measurement, the treated cells were stained with 5μg/mL JC-1 (Invitrogen) in the dark for 15 min. Cells were then washedwith PBS once, followed by FACS analysis. Photomultiplier settings wereadjusted to detect green fluorescence (λ_(em)=525 nm) of JC-1 monomerusing filter 1 (FL-1 detector) and the red fluorescence (λ_(em)=590 nm)of JC-1 aggregates using filter 2 (FL2 detector). The ratio of JC-1aggregate/monomer (red/green or FL2/FL1) is indicative of the membranepotential. Data was collected from normal cell populations for eachsample, which was gated according to the non-treatment controls based onforward and side scatters. (C) For ROS measurement, L40 treated cellswere stained with 2 μg/mL of dehydrorhodamine 123 for 30 min before FACSanalysis. In each experiment above, at least 10,000 events wereanalyzed. The data represents the average of a duplica-experiment.

FIG. 9 shows that only low doses of rapamycin (below the growthinhibition doses) prevent the loss of senescent WI-38 cells. (A) WI-38cells were treated with the indicated concentrations of rapamycin as inFIG. 7. The treatment started at passage 29. Cells entered senescence atpassage 31. After 65-days in senescence, surviving cells were measuredby MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, atetrazole) staining. The purple color of MTT generated by mitochondrialreductase was read in 570 nm by a microplate reader. The data is arepresentative of three independent duplica-experiments. (B) The effectof rapamycin on growth of WI-38 cells. Cells were cultured as in FIG. 7.Arrows indicate the time point when rapamycin was added. 25 pM rapamycinhas little effect on growth rate, but increases the population doubling(PD) from 5.18 to 6.82. Population doublings were calculated using thefollowing formula: PD=log(N_(f)/N₀)/log2, where N_(f) is the final cellnumber and N₀ is the number of initially seeded cells. Data is arepresentative of two independent duplica-experiments. (C) The low dosesof rapamycin increase protein levels of p53, p21 and pRB. WI-38 cells onthe 20^(th) day after the last split (senescence) were treated withrapamycin for 18 hrs. Cell lysates were then analyzed by Westernblotting using antibodies specifically against p53, p21 or pRB.

FIG. 10 shows that mitochondrial mass is increased in humanlymphoblastoid L40 by a 2-day treatment with (A) 10 μM LY294002 (a PI3Kinhibitor), 2 μM diallyl trisulfide (DATS), 1 μM benzyl isothiocyanate(BITC), 1 μM phenyl isothiocyanate (PITC), 2 μg/mL resveratrol (RSV) and0.03 μM lycopene, and (B) with 6.7 μM PEITC, 5 mM silibinin, 1.25 mMselenite, 2.5 mM genistein, 250 μg/mL grape seed extract (GSE), 50 μg/mLEGCG, 3 mg/mL bilberry extract (BE), 1 μM AITC 50 pM rapamycin 250 μMAICAR, and reduced glucose (from 0.4% to 0.2%). Mitochondrial mass wasmonitored by staining the 60% Ethanol-fixed cells with MitoTracker GreenFM prior to FACS analysis.

FIG. 11 shows that a number of chemopreventive agents or anti-agingagents inhibited cell death triggered by inactivation of cdc13-1p. The6.7 μM PEITC, 5 mM silibinin, 1.25 mM selenite, 2.5 mM genistein, 250μg/mL grape seed extract (GSE), 50 μg/mL EGCG, and 3 mg/mL bilberryextract (BE) were incubated with cells at 37° C. for about 30 hrs. Thecell survival was measured by the colony formation assay at a permissivetemperature 24° C.

FIG. 12 shows that the low doses of rapamycin and AICAR reverse themitochondrial mass decrease induced by 12-O-tetradecanoylphorbol13-acetate (TPA). NIH3T3 cells were incubated in DMEM medium (DMEM with10% FCS, 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mMglutamine) in the presence of control DMSO, 10 μM TPA, 1 nM rapamycin,10 μM TPA+ 1 nM rapamycin, 40 μM AICAR, or 10 μM TPA+ 40 μM AICAR at 37°C. in a humidified atmosphere consisting of 95% air and 5% CO₂ for twodays. Cells were then harvested by trypsin and fixed by 60% ethanol. Thecells were stained with MitoTracker Greeen FM in darkness for 30 minprior to FACS analysis. The data represents the average of atriplica-experiment.

FIG. 13 shows that the low doses of rapamycin and AICAR prevent NIH3T3tumor transformation induced by TPA. (A) TPA (10 μM) was incubated withNIH3T3 cells in 0.39% soft agar in the presence of DMSO, 1 nM rapamycin,or 250 nM AICAR, and cultured for 7 days prior to counting coloniesunder a microscope. (B) The data represents the average of fourexperiments.

FIG. 14 shows that rapamycin at low doses, 0.2 and 2 pM, extends thelife span of cultured CGN cells (A) and reduces ROS levels in CGN cells(B). Cerebellar granule neuron (CGN) cultures were prepared from7-day-old rat pups. Briefly, the cerebellum was removed from the brain,minced into fine pieces, trypsinized at 37° C. for 15 min, filteredthrough a 40-μM mesh, and pelleted by centrifugation. The pelletscontaining cerebellar granule neurons were resuspended in B27supplemented neurobasal medium containing 25 mM KCl. For their life spanin culture, cells were then seeded into a 24-well plate (1plate/cerebellum) and cultured in Neurobasal medium (Invitrogen)supplemented with B27, 20 mM KCl, 0.5 mM Glutamine, 100 units/mLpenicillin, 100 μg/mL streptomycin). Rapamycin was added 7 days after inplate. 31 days later, survival of neuron cells was determined using aMTT assay (A). For ROS analysis, fresh isolated CGN cells in suspensionculture in Neurobal complete medium were seeded in 12×75 mm tubes at adensity of 1 million cells/mL/tube. The cells were treated withrapamycin for 20 hrs and then stained with 2 μg/mL dehydrorhodamine 123for 30 min prior to FACS analysis (B).

FIG. 15 shows that the low doses of rapamycin reduce the braininfarction volume in a rat model of stroke. (A) Rapamycin (10 μg/kg)reduced the brain damage. Middle cerebral artery (MCA) occlusion modelof ischemic stroke was used. SHR-SP rats were randomly divided into twogroups (n=8 in each group): a matched control DMSO group and rapamycingroup. Rapamycin and the control DMSO were administrated 10 minutesafter MCA occlusion. Brain samples were harvested 24 h after MCAocclusion. The coronal sections (2 mm in thickness) were immediatelystained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). Theinfarction region containing dead cells can not be stained as pale,while the normal region with living cells as red. The infarction areaand hemisphere areas of each section (both sides) were traced andquantified by an image analysis system (Microsystems Type DM LB2, Leica,Germany). The possible interference of a brain edema in assessing theinfarction volume was corrected for with a standard method ofsubtracting the volume of the nonischemic ipsilateral hemisphere fromthe contralateral hemisphere volume. The infarction area was expressedas a percentage of the contralateral hemisphere. (B) The low doses ofrapamycin prevent brain damages induced by ischemic infarction.Rapamycin at 0, 0.3, 1, 3 and 10 μg/kg was administrated to SHR-SP rats(n=8 in each group) for 20 days prior to MCA occlusion.

FIG. 16 shows that low doses of rapamycin decrease ROS levels induced byMPP⁺ in human primary fibroblast WI-38 cells (200 μM MPP⁺ was used).MPP+ with various concentrations of rapamycin was incubated with WI-38cells for 3 days. Cells were stained with dehydrorhodamine 123 in thedark for 30 min prior to FACS analysis.

FIG. 17 shows that the low dose of rapamycin at 10 μg/kg, but not at 100μg/kg, reduces the myocardial infarction (MI) volume in a rat model.Male Sprague-Dawley (SD) rats of 200 to 250 g were used (n=10-12 foreach group). Rapamycin at doses of 0, 10, and 100 μg/kg/day wasadministrated for 3 days prior to the MI experiment. Under etheranesthesia, the heart was exteriorized, and the left anterior descendingarteries were ligatured between the pulmonary outflow tract and the leftatrium. Then the beating heart was quickly returned to its normalposition, the thorax was closed, and the air was removed. The rats werereturned to the cages. Five hours after the coronary artery ligature,the rats were sacrificed. The left ventricle was isolated and cut into 4to 5 slices perpendicular to the cardiac long axis. The slices werestained in nitro blue tetrazolium phosphate buffer. The normal tissuewas stained in blue, while necrotic tissue remained unstained. Thestained and unstained tissues were isolated and weighed separately. TheMI size was expressed as a fraction of the total left ventricularweight.

FIG. 18 shows a model of glucose or TOR regulates aging processesthrough the AMPK/ROS/mitochondria pathway in various tissues, which leadto age-related disorders.

FIG. 19 illustrates a method for high-throughput screening to identifyand to detect anti-aging candidates using cell surviving assays inyeast.

FIG. 20 illustrates a method for high-throughput screening to identifyand to detect anti-aging candidates using the ROS assay in yeast.

FIG. 21 shows examples of detection of an anti-aging agent using theyeast mutant cdc17-1, or cdc17-2. The mutant cells were diluted into afresh YEPD medium containing rapamycin of 0, 1 and 3 nM, or 0.5% glucoseYEPD medium and incubated at about 37° C. for 22 hrs. Cells were thenserially diluted 10-fold and spotted on a YEPD plate. The plate wasincubated at a permissive temperature 24° C. for colony forming fromsurviving cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes novel methods for identifying,detecting, and purifying anti-aging agents and use of the agents for theprevention and treatment of age-related diseases or disorders. Theinvention is based on, inter alia, the following discoveries: (1) thatinhibition of nutrient signals prolongs the cell cycle-arrested stateinduced by telomere dysfunction in yeast via the AMPK and subsequentmitochondrial pathway; (2) that low doses of rapamycin, glucoserestriction, and AMPK activators stimulate mitochondrial function andprolong senescence in primary human fibroblasts; (3) that severalanti-aging and cancer chemopreventive agents stimulate mitochondrialfunction and inhibit loss of senescent cells in both yeast and humancells; (4) that many age-related diseases or disorders are associatedwith dysfunction of mitochondria and/or telomeres; and (5) that lowdoses of rapamycin prevent cerebellar and myocardial ischemicinfarction, reduce ROS triggered by MPP+, increase life span of culturedprimary neuron cells and inhibit tumor cell transformation.

In a first aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions against a senescence model system andmonitoring their anti-aging activity.

In one embodiment of the first aspect, the present invention provides amethod of identifying or detecting an agent for preventing or treatingan age-related disease or disorder, wherein the anti-aging activity ispreventing deterioration of a cell cycle-arrested state in senescent orpost-mitotic cells.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the anti-agingactivity is stimulating, improving, or maintaining mitochondrialfunction.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the anti-agingactivity is preventing an age-related disease or disorder that isassociated with loss of mitochondrial or telomere function.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the anti-agingactivity is preventing increase of reactive oxygen species (ROS) orapoptotic death induced by telomere dysfunction.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a mutant yeast comprising a dysfunctional telomere.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a primary human cell line that exhibits insufficienttelomerase activity.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a human cell line that exhibits telomere dysfunctioncaused by a mutation or defect in a telomere binding protein ortelomerase.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a telomere dysfunction model caused by a chemical agent.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a model created by oncogene activation and/or activationof DNA damage response.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a cell line of mouse, rat or S. Pombe which exhibitstelomere dysfunction.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, the method comprising thesteps of:

-   -   (i) incubating a compound or composition with yeast cells under        conditions where a cell cycle is arrested by telomere        dysfunction or DNA damage;    -   (ii) measuring the population of dead yeast cells using an        apoptotic assay, or alternatively;    -   (iii) removing the conditions under which the cell cycle is        arrested and measuring the number of survived cells; and    -   (iv) comparing the population of dead cells obtained in        step (ii) or the number of survived cells obtained in step (iii)        with the population of dead cells or the number of survived        cells, respectively, obtained in a control experiment under the        same conditions as in step (i) but in the absence of said        compound or composition,    -   wherein a decreased population of the dead yeast cells obtained        in step (ii) or an increased number of the survived cells        obtained in step (iii) as compared with those of the control        experiment would indicate that the compound or composition        incubated is a candidate of anti-aging agent.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, the method comprising thesteps of:

-   -   (i) incubating a compound or composition with mammalian        senescent cells for a period of time;    -   (ii) measuring the population of survived senescent cells; and    -   (iii) comparing the population of the survived mammalian        senescent cells of step (vi) with population of survived        senescent cells obtained in a control experiment,    -   wherein an increased population of the survived senescent cells        as compared to the control experiment would indicate that the        compound or composition incubated with the cells is a candidate        of anti-aging agent.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, the method comprising thesteps of:

-   -   (i) incubating a compound or composition with normal growing        human cells for a period of time;    -   (ii) measuring mitochondrial biogenesis of the human cells by        measuring mitochondrial mass, mitochondrial DNA content, or        expression of mitochondrial transcription factors; and    -   (iii) comparing the result of step (ix) with that of a control        experiment,    -   wherein an enhanced mitochondrial biogenesis obtained in        step (ix) as compared to the control experiment would indicate        that the identified compound or composition is a candidate of        anti-aging agent.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, the method comprising anycombination of the steps described in the above three embodiments.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a mutant yeast comprising a dysfunctional telomereselected from cdc13-1, cdc13-2, stn1-1, cdc17-1, cdc17-2, hdf1, hdf2,est1, est2, and est3.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a primary human cell line comprising at least one offibroblasts, endothelial cells, and epithelial cells, which exhibitsinsufficient telomerase activity.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a human cell line comprising a mutation in TRF2, POT1,TERT, TERC, or WRN, which exhibits telomere dysfunction.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the senescencemodel system is a telomere dysfunction model caused by a chemical agentselected from the group consisting of bleomycin, adriamycin, andG-quadruplex ligands.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the age-relateddisease or disorder is an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder.

In another embodiment of the first aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said one or morecompounds or compositions each belong to a library of compounds and/orcompositions. Thus, a preferred embodiment of this invention encompassesa high throughput screening of a plurality of compounds or compositions.

In a second aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and measuring their activity against atleast one of components of the TOR/AMPK/Mitochondria/Senescence pathway,wherein the agent (a) extends replication potential, (b) maintainssenescence or cell cycle-arrested state in post-mitotic cells, or (c)prevents deterioration of mitochondria or cell death followingdeterioration of senescence or cell cycle arrested-state. This aspect isrelated to the first aspect of the present invention in that saidanti-aging activity is on any of the components of theTOR/AMPK/Mitochondria/Senescence pathway with an effect of maintainingsenescence.

In one embodiment of the second aspect, the present invention provides amethod of identifying or detecting an agent for preventing or treatingan age-related disease or disorder, wherein the age-related disease ordisorder is associated with senescence deterioration, cell deathfollowing deterioration of a cell cycle-arrested state in senescent andpost-mitotic cells, accelerated mitochondrial deterioration andincreased oxidative stress, or telomere dysfunction.

In another embodiment of the second aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said components ofthe TOR/AMPK/Mitochondria/Senescence pathway comprise Insulin/IGF,Insulin/IGF receptors, PI3K, PDK1, PTEN, TSC1, TSC2, AKT, Rheb, raptor,GβL, S6K, TOR, AMPK, STRAD, MO25, LKB1, glucose uptake, amino aciduptake, CaMKKβ, PGC-1α, PGC-1β, NRF-1, NRF-2, TFAM, TFB1M, TFB2M, ERRs(e.g., ERRα, ERRβ and ERRγ), PPARs (e.g., PPARα, PPARδ and PPARγ),SIRT1, RIP140, PRC, POLRMT, ATM, p53, p21, p19^(ARF), WAF1, P16^(INK4a),pRB, E2F, PTEN, and p27^(KIP1).

In another embodiment of the second aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the age-relateddisease or disorder is an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder.

In another embodiment of the second aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said one or morecompounds or compositions each belong to a library of compounds and/orcompositions.

In a third aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the mitochondrial biogenesis pathway, whereinthe agent (a) extends replication potential, (b) maintains senescence orcell cycle-arrested state in post-mitotic cells, or (c) preventsdeterioration of mitochondria or cell death following senescencedeterioration. This aspect is related to the first aspect of the presentinvention in that said anti-aging activity is against any of thecomponents of the mitochondrial biogenesis pathway with an effect ofmaintaining senescence.

In one embodiment of the third aspect, the present invention provides amethod of identifying or detecting an agent for preventing or treatingan age-related disease or disorder, wherein the age-related disease ordisorder is associated with cell death following deterioration of a cellcycle-arrested state in senescent and post-mitotic cells, senescencedeterioration, accelerated mitochondrial deterioration and increasedoxidative stress, or telomere dysfunction, and.

In another embodiment of the third aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the components ofthe mitochondrial biogenesis pathway comprise AMPK, STRAD, MO25, LKB1,CaMKKβ, PGC-1α, PGC-1β, NRF-1, NRF-2, TFAM, TFB1M, TFB2M, ERRs (e.g.,ERRα, ERRβ and ERRγ), PPARs (e.g., PPARα, PPARδ and PPARγ), SIRT1,RIP140, PRC, and POLRMT.

In another embodiment of the third aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the age-relateddisease or disorder is an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder.

In another embodiment of the third aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said one or morecompounds or compositions each belong to a library of compounds and/orcompositions.

In a fourth aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating aging or anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the AMPK pathway, wherein the agent (a)extends replication potential, (b) maintains senescence or cellcycle-arrested state in post-mitotic cells, or (c) preventsdeterioration of mitochondria or cell death following senescencedeterioration. This aspect is related to the first aspect of the presentinvention in that said anti-aging activity is against any of thecomponents of the AMPK pathway with an effect of maintaining senescence.

In one embodiment of the fourth aspect, the present invention provides amethod of identifying or detecting an agent for preventing or treatingan age-related disease or disorder, wherein the age-related disease ordisorder is associated with senescence deterioration, age-related cellloss, or tumorigenesis and malignant progression of cancers.

In another embodiment of the fourth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the components ofthe AMPK pathway comprise AMPK, ATM, LKB1, STRAD, MO25, and CaMKKβ.

In another embodiment of the fourth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the age-relateddisease or disorder is an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder.

In another embodiment of the fourth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said one or morecompounds or compositions each belong to a library of compounds and/orcompositions.

In a fifth aspect, the present invention provides a method ofidentifying or detecting an agent for preventing or treating anage-related disease or disorder, the method comprising screening one ormore compounds or compositions and detecting their activity against atleast one of components of the senescence pathway, wherein the agentmaintains senescence or cell cycle-arrested state in post-mitotic cellsor prevents deterioration of mitochondria or cell death followingsenescence deterioration. This aspect is related to the first aspect ofthe present invention in that said anti-aging activity is against any ofthe components of the senescence pathway with an effect of maintainingsenescence.

In one embodiment of the fifth aspect, the present invention provides amethod of identifying or detecting an agent for preventing or treatingan age-related disease or disorder, wherein the age-related disease ordisorder is associated with senescence deterioration, cell deathfollowing deterioration of a cell cycle-arrested state in senescent andpost-mitotic cells, accelerated mitochondrial deterioration andincreased oxidative stress, or telomere dysfunction.

In another embodiment of the fifth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the components ofthe senescence pathway comprise ATM, p53, p21, p19^(ARF), WAF1,p16^(INK4a), pRB, E2F, PTEN, and p27^(KIP1).

In another embodiment of the fifth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein the age-relateddisease or disorder is an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder.

In another embodiment of the fifth aspect, the present inventionprovides a method of identifying or detecting an agent for preventing ortreating an age-related disease or disorder, wherein said one or morecompounds or compositions each belong to a library of compounds and/orcompositions.

In a sixth aspect, the present invention provides a method of preventingor treating an age-related disease or disorder, the method comprisingadministering to a subject in need thereof a composition comprising anagent identified according to any embodiments in the first to fifthaspects of the present invention as described above, or apharmaceutically acceptable salt, solvate, or prodrug thereof. Thisaspect thus encompasses the use of any of the anti-aging compounds orcompositions identified by the methods described herein for preparationor manufacture of a medicament for the prevention or treatment of anage-related disease or disorder as encompassed by this disclosure.

In one embodiment of the sixth aspect, the present invention provides amethod of preventing or treating an age-related disease or disorder,wherein the age-related disease or disorder is associated withdeterioration of a cell cycle-arrested state in senescent orpost-mitotic cells, mitochondrial dysfunction, or telomere dysfunction.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the agent is selected from organic molecules,inorganic molecules, natural products, peptides, proteins, DNAs, RNAs,and metabolic intermediates thereof.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the agent is selected from AICAR, low dose ofrapamycin or analogs thereof, EGCG, grape seed extract, bilberryextract, selenite, genistein, diallyl trisulfide, benzyl isothiocyanate,phenyl isothiocyanate, phenethyl isothiocyanate, resveratrol, lycopene,and allyl isothiocyanate.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises a second agentselected from an antioxidant, an antihypertensive agent, alipid-lowering agent, an anti-stroke agent, an anti-cancer agent, and adifferent anti-aging agent.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises an antioxidantselected from vitamin C, vitamin E, beta carotene and other carotenoids,selenium, lipoic acid, lycopine, lutein, zeaxanthin, coenzyme Q10,glutathione, N-acetyl cysteine, melatonin, genistein, estrodiol, teaextract, and grape seed extract.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises a pharmaceuticallyacceptable carrier.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition is administered orally, parenterally,topically, transdermally, or in a suppository or aerosol form.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is an abnormalproliferative disease, a degenerative disease, or a function-decreasingdisorder.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is selected fromtumorigenesis and malignant cancer development, neurodegeneratingdisease, myocardial infarction (heart attack), heart failure,atherosclerosis, hypertension, osteoarthritis, osteoporosis, sarcopenia,loss of bone marrow, cataract, multiple sclerosis, Sjogren, Rheumatoidarthritis, degraded immune function, diabetes, Idiopathic pulmonaryfibrosis, and age-related macular degeneration, cerebellar infarction,stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease,and disorders caused by the decline in testosterone, estrogen, growthhormone, IGF-I, or energy production.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said subject is a mammal.

In another embodiment of the sixth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said subject is a human.

In a seventh aspect, the present invention provides a method ofpreventing or treating an age-related disease or disorder, the methodcomprising administering to a subject in need thereof a compositioncomprising an AMPK activator, or a pharmaceutically acceptable salt,solvate, or prodrug thereof, which directly or indirectly activatesAMPK, increases mitochondrial biogenesis, and maintains a cellcycle-arrested state in the senescent or post-mitotic cells of thesubject. This aspect of the invention thus encompasses the use of anAMPK activator for preparation or manufacture of a medicament for theprevention or treatment of an age-related disease or disorderencompassed by this disclosure. This aspect is related to the sixthaspect of the present invention in that said agent is an AMPK activator.

In one embodiment of the seventh aspect, the present invention providesa method of preventing or treating an age-related disease or disorder,wherein the age-related disease or disorder is associated withmitochondrial function loss, telomere dysfunction, senescencedeterioration and age-dependent cell loss, or mitochondrialdeterioration or cell cycle-arrested state in post-mitotic cells.

In one embodiment of the seventh aspect, the present invention providesa method of preventing or treating an age-related disease or disorder,wherein said AMPK activator is selected from AICAR, metformin,2-deoxyglucose, 3-O-methylglucose, LY294002, berberine, phenformin,A-769662, thiazolidinediones, dexamethasone, statins, leptin,adiponectin, cilostazol, EGCG, senelite, allyl isothiocyanate, andphenethyl isothiocyanate.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises a second agentselected from an antioxidant, an antihypertensive agent, alipid-lowering agent, an anti-stroke agent, an anti-cancer agent, and adifferent anti-aging agent.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises a pharmaceuticallyacceptable carrier.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition is administered orally, parenterally,topically, transdermally, or in a suppository or aerosol form.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is an abnormalproliferative disease, a degenerative disease, or a function-decreasingdisorder.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is selected fromtumorigenesis and malignant cancer development, neurodegeneratingdisease, myocardial infarction (heart attack), heart failure,atherosclerosis, hypertension, osteoarthritis, osteoporosis, sarcopenia,loss of bone marrow, cataract, multiple sclerosis, Sjogren, Rheumatoidarthritis, degraded immune function, diabetes, Idiopathic pulmonaryfibrosis, and age-related macular degeneration, cerebellar infarction,stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease,and disorders caused by the decline in testosterone, estrogen, growthhormone, IGF-I, or energy production.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said subject is a mammalian animal.

In another embodiment of the seventh aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said subject is a human.

In an eighth aspect, the present invention provides a method ofpreventing or treating an age-related disease or disorder, the methodcomprising administering to a subject in need thereof a compositioncomprising a target-of-rapamycin (TOR) inhibitor, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, wherein the TOR inhibitor(a) extends replication potential, (b) maintains senescence or cellcycle-arrested state in post-mitotic cells, or (c) preventsdeterioration of mitochondria or cell death following senescencedeterioration. This aspect of the invention thus encompasses the use ofa TOR inhibitor, or a pharmaceutically acceptable salt, solvate, orprodrug thereof, for preparation or manufacture of a medicament for theprevention or treatment of an age-related disease or disorderencompassed by this disclosure. This aspect is related to the sixthaspect of the present invention in that said agent is a TOR inhibitor.

In one embodiment of the eighth aspect, the present invention provides amethod of preventing or treating an age-related disease or disorder,wherein the age-related disease or disorder is associated withmitochondrial function loss, telomere dysfunction, senescencedeterioration and age-dependent cell loss, or mitochondrialdeterioration or cell cycle-arrested state in post-mitotic cells.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said TOR inhibitor is a low dose of rapamycin or ananalog thereof.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said TOR inhibitor is a low dose of rapamycin or ananalog selected from Deforolimus, AP-23675, AP-23841, Zotarolimus,CCI779/Temsirolimus, RAD-001/Everolimus, 7-epi-rapamycin,7-thiomethyl-rapamycin, 7-epi-trimethoxy-rapamycin,2-desmethyl-rapamycin, and 42-O-(2-hydroxy)ethyl-rapamycin, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said TOR inhibitor is a low dose of rapamycin, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In another embodiment of the eighth aspect, the TOR inhibitor rapamycinis used at low doses from about 0.1 to about 10000 pM in serum medium,or from 0.1 to about 10000 ng/kg/day in animal. It is a surprisingdiscovery that rapamycin at these low doses exhibits novel functionrather than inhibits protein cell growth, although it is traditionallyknown that rapamycin is an immunosuppressant at therapeutic doses (1mg/day to 5 mg/day) which inhibit cell growth at G1 phase of cell cycleand may also target other functional protein complexes. As a result, thetherapeutic doses of rapamycin thus show various side effects, includingincreases in serum cholesterol and triglycerides, renal functiondeficiency, anemia, impaired wound healing, diarrhea, asthenia,hypotension, pain, malignant neoplasm progression, hepatic neoplasmmalignant, ascites, failure to thrive, mental status changes, splenicinfarction, and colitis, etc. These side effects can be avoided when lowdoses of rapamycin are used according to the present invention.

The effective dosage of rapamycin or its analogs may vary depending uponthe particular compound utilized, the mode of administration, thespecific disorders being treated, as well as various physical factorsrelated to the individual being treated. As used in accordance with thisinvention, satisfactory results may be obtained when rapamycin isadministered at a daily oral dosage of about 0.01 to about 50 μg/daydepending on the specific tissue disease or disorder treated, which isestimated to be about 0.001% to about 5% of the therapeutic doses (1mg/day to 5 mg/day). As used in accordance with this invention, theadministration schedule of two to four days with rapamycin followed bytwo to five days of rapamycin-free may yield best results with the leastadverse effects.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said low doses of rapamycin, or an analog thereof, arebelow about 10%, below about 8%, below about 6%, below about 4%, belowabout 2%, below about 1%, below about 0.1%, below about 0.01%, or belowabout 0.001% of an approved therapeutic dose.

In one preferred embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein rapamycin, or an analog thereof, is administered at adose in the range of about 8% to about 10% of an approved therapeuticdose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 6% to about 8% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 4% to about 6% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 2% to about 4% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 1% to about 2% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 0.1% to about 1% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 0.01% to about 0.1% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 0.01% to about 0.1% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 0.001% to about 0.01% of anapproved therapeutic dose.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein rapamycin, or an analog thereof, isadministered at a dose in the range of about 0.0001% to about 0.001% ofan approved therapeutic dose.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the low dose of rapamycin is administered as anisolated compound.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the low dose of rapamycin is administered as a crudeextract.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the low dose of rapamycin is administered as anunpurified microorganism comprising rapamycin.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the low dose of rapamycin is administered as anunpurified microorganism Streptomyces hygroscopicus, which comprisesrapamycin.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises a second agentselected from an antioxidant, an antihypertensive agent, alipid-lowering agent, an anti-stroke agent, an anti-cancer agent, and adifferent anti-aging agent.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises an antioxidant tocontrol ROS from both cellular and mitochondrial levels.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition further comprises an antioxidantselected from vitamin C, vitamin E, beta carotene and other carotenoids,selenium, lipoic acid, lycopine, lutein, zeaxanthin, coenzyme Q10,glutathione, N-acetyl cysteine, melatonin, genistein, estrodiol, teaextract, and grape seed extract.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the composition is administered orally, parenterally,topically, transdermally, or in a suppository or aerosol form.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is an abnormalproliferative disease, a degenerative disease, or a function-decreasingdisorder.

In another embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein the age-related disease or disorder is selected fromtumorigenesis and malignant cancer development, neurodegeneratingdisease, myocardial infarction (heart attack), heart failure,atherosclerosis, hypertension, osteoarthritis, osteoporosis, sarcopenia,loss of bone marrow, cataract, multiple sclerosis, Sjogren, Rheumatoidarthritis, degraded immune function, diabetes, Idiopathic pulmonaryfibrosis, and age-related macular degeneration, cerebellar infarction,stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease,and disorders caused by the decline in testosterone, estrogen, growthhormone, IGF-I, or energy production.

In one preferred embodiment of the eighth aspect, the present inventionprovides a method of preventing or treating an age-related disease ordisorder, wherein said agent is a lose dose of rapamycin or an analogthereof, and wherein the age-related disease or disorder is selectedfrom tumorigenesis and malignant cancer development, neurodegeneratingdisease, myocardial infarction (heart attack), heart failure,atherosclerosis, hypertension, osteoarthritis, osteoporosis, sarcopenia,loss of bone marrow, cataract, multiple sclerosis, Sjogren, Rheumatoidarthritis, degraded immune function, diabetes, Idiopathic pulmonaryfibrosis, and age-related macular degeneration, cerebellar infarction,stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease,and disorders caused by the decline in testosterone, estrogen, growthhormone, IGF-I, or energy production.

In another preferred embodiment of the eighth aspect, the presentinvention provides a method of preventing or treating an age-relateddisease or disorder, wherein said agent is a low dose of rapamycin or ananalog thereof, and wherein the age-related disease or disorder isselected from tumorigenesis or malignant progression of a cancer,Parkinson's disease, stroke, cerebellar infarction, and myocardialinfarction.

In another preferred embodiment of the eighth aspect, the presentinvention provides the use of rapamycin at low concentrations inprolonging senescence induced by age-dependent telomere dysfunction,oncogene activation, or DNA damaging agents (e.g., ROS, anticancerdrugs, UV or ionizing irradiation), since the same senescence mechanismis involved in these processes, via the DNA damage response. Thus, themethods of the present invention may be used to treat various benigntumors and prevent them from malignancy progression. The methods of thepresent invention may thus be used in populations that have high risk ofcancers, such as aged populations, and people who are often in contactwith mutagens, or subject to UV, or ionizing irradiations. The methodsof the present invention may also be used in patients who take drugswith high risks of inducing cancers, such as women who are takinghormone replacements. Furthermore, the methods of the present inventionmay also be used in cancer patients who are undergoing a chemotherapythat could induce secondary cancers.

In another preferred embodiment of the eighth aspect, the presentinvention provides the use of rapamycin at low concentrations inpreventing brain damage induced by stroke. Thus, a low dose of rapamycincan be used as the emergency drug to treat stroke, including bothischemic and hemorrhagic stroke. To yield the best result, the presentinvention also provides the use of a low dose of rapamycin incombination of the emergency medicine to treat ischemic stroke such astissue plasminogen activator (t-PA), a clot-dissolving medicine.

In another preferred embodiment of the eighth aspect, the presentinvention provides the use of rapamycin at low concentrations inpreventing stroke and reoccurring strokes. In order to have the bestprevention results, in another embodiment of the method, the presentinvention also provides the use of low doses of rapamycin in combinationwith medicines of different mechanisms that reduce the risk of stroke.These medicines include, but are not limited to, antiplatelet medicines(e.g., clopidogrel, Agrrenox), anticoagulants (e.g., warfarin, heparin),lipid lowering drugs (e.g., statins) and blood pressure medicines (e.g.,Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II receptorblockers (ARBs), Beta-blockers, Diuretics, and Calcium channelblockers).

In another preferred embodiment of the eighth aspect, the presentinvention provides the use of rapamycin at low concentrations inpreventing neuron degenerative diseases, including, but not limited to,Alzheimer's, Parkinson's, Huntington's diseases.

In another preferred embodiment of the eighth aspect, the presentinvention provides the use of rapamycin at low concentrations inpreventing damages at heart induced by myocardial infarction. Thus, theinvention provides the use of rapamycin at a low dose in preventingmyocardial infarction or heart attack. In order to have best result, therapamycin can also be used in combination with medicines of differentmechanisms for preventing the risk of heart attack. These medicinesinclude, but are not limited to, antiplatelet medicines (e.g.,clopidogrel, Agrrenox), anticoagulants (e.g., warfarin, heparin), lipidlowering drugs (e.g., statins), blood pressure medicines (e.g.,Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II receptorblockers (ARBs), Beta-blockers, Diuretics, and Calcium channelblockers), and blood sugar control medicines (e.g., Metformin andPioglitazone).

A person of skill in the art would understand that the distribution ofrapamycin is uniform in different tissue or cell type, and each compoundor composition identified from the anti-aging screen may have a specificdistribution pattern in different tissues or cell types. In one aspectof the method, the invention provides the use of rapamycin at a low dosein combination with at least one other anti-aging agent, including, butnot limited to AICAR, 2-deoxyglucose, LY294002, metformin, EGCG, GSE,senelite, genistein, silibinin, allyl isothiocyanate, phenethylisothiocyanate, bilberry extracts, diallyl trisulfide, benzylisothiocyanate, resveratrol, and lycopene.

In another embodiment of the eighth aspect, rapamycin at a low dose maybe administered in any useful manner, including oral, via implants,parenteral (including intravenous, intraperitoneal and subcutaneousinjections), topical, rectal, intranasal, vaginally, inhaled, aerosol,and transdermal forms. The transdermal administrations include alladministrations across the surface of the body and the inner linings ofbodily passages including the epithelial and mucosal tissues. Suchadministrations may be carried out using the present compounds orpharmaceutically acceptable salts thereof in lotions, creams, foams,patches, suspensions, solutions, and suppositories (rectal and vaginal).

In another embodiment of the eighth aspect, the present inventionprovides the use of rapamycin at a low dose in the form of an ingredientof solid food, beverages or liquid foods, including alcoholic ornon-alcoholic ones, for example, water, wine, and juices, etc.

In a ninth aspect, the present invention provides a method for detectingan anti-aging agent in a biological sample, the method comprising thesteps of:

-   -   (i) optionally diluting the biological sample with a solvent;    -   (ii) incubating the diluted sample with mutant yeast cells under        conditions where the cell cycle of the yeast cells is arrested        by telomere dysfunction or DNA damage;    -   (iii) removing the conditions under which the cell cycle is        arrested and measuring the number of survived yeast cells; and    -   (iv) comparing the number of survived cells obtained in        step (iii) to the number of survived cells in a control        experiment under the same conditions as those of the incubating        step (ii) except for the absence of the subject biological        sample,    -   wherein the increased number of survived cells obtained in        step (iii) compared to the number of survived cells in the        control experiment indicates that the biological sample        comprises an anti-aging agent.

In a tenth aspect, the present invention provides a method fordetermining biological concentration of an anti-aging agent in abiological sample, the method comprising the steps of:

-   -   (i) optionally diluting a subject biological sample with a        solvent;    -   (ii) incubating the diluted biological sample with mutant yeast        cells under conditions where the cell cycle of the yeast cells        is arrested by telomere dysfunction or DNA damage;    -   (iii) removing the conditions under which the cell cycle is        arrested and measuring the number of survived yeast cells;    -   (iv) optionally comparing the number of survived cells obtained        in step (iii) to the number of survived cells in a control        experiment under the same conditions as those of the incubating        step (ii) except for the absence of the subject biological        sample; and    -   (v) calculating the biological concentration of the anti-aging        agent by applying the number of survived yeast cells to a        pre-established standard equation or curve between the        concentration of anti-aging agent and the number of survived        yeast cells.

In one embodiment of the tenth aspect, the present invention provides amethod to prepare a standard equation or curve to be used forcalculation of biological concentration of an anti-aging agent in abiological sample, the method comprising the steps of:

-   -   (vi) preparing a plurality of standard solutions having        different known concentrations of a purified anti-aging agent        using a solvent to be used for incubating the subject biological        sample;    -   (vii) incubating the standard solutions with the mutant yeast        cells under conditions where the cell cycle is arrested by        telomere dysfunction or DNA damage;    -   (viii) removing the conditions under which the cell cycle is        arrested and measuring the number of survived yeast cells in        each incubated standard solution; and    -   (ix) plotting the numbers of survived cells obtained in        step (viii) against the corresponding concentrations of the        anti-aging agent to obtain a standard curve and/or to obtain a        standard equation.

This aspect is related to the ninth aspect of the present invention inthat a quantitative analysis is involved by calculating the biologicalconcentration of an anti-aging agent using a pre-established equation orcurve described above. Thus, the present invention encompasses methodscomprising any reasonable combinations of the steps described in the twoaspects.

In another embodiment of the tenth aspect, the present inventionprovides a method for determining biological concentration of ananti-aging agent in a biological sample, wherein the anti-aging agent isa compound or composition identified or used according to any of theembodiments in any aspects of the present invention as described above.

In another embodiment of the tenth aspect, the present inventionprovides a method for determining biological concentration of ananti-aging agent in a biological sample, wherein the anti-aging agent israpamycin or an analog thereof. In a preferred embodiment, the rapamycinor analog is at a low dose.

In another embodiment of the tenth aspect, the present inventionprovides a method for determining biological concentration of ananti-aging agent in a biological sample, wherein the mutant yeast isselected from cdc13-1, cdc13-2, stn1-1, cdc17-1, cdc17-2, hdf1, hdf2,est1, est2, and est3.

Other aspects or preferred embodiments of the present invention mayinclude any suitable combinations of the embodiments disclosed herein.Yet other aspects and embodiments may be found in other parts of thedescription provided herein.

Definitions

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

As used herein, the term “optional” or “optionally” means that thesubsequently described event or circumstance can occur or otherwise, andthat the description includes instances where the event or circumstanceoccurs and instances where it does not.

As used herein, the term “age-related disorder” or “age-related disease”refers to disorders or diseases in which aging is a major risk factor.Based on the type of diseases, age-related diseases or disorders includethree main types: (1) abnormal poliferative diseases, such as cancer;(2) degenerative diseases, including neuron degenerating disease(Alzheimer's, Parkinson's, stroke), myocardial infarction, heartfailure, atherosclerosis, hypertension, osteoarthritis, osteoporosis,sarcopenia, loss of bone marrow, Rheumatoid arthritis, degraded immunefunction, diabetes, Idiopathic pulmonary fibrosis, age-related maculardegeneration; and (3) function decreasing disorders, including declinesin testosterone, estrogen, growth hormone, IGF-I, reduced energyproduction and so on. Based on the type of cells involved, age-relateddiseases or disorders can also be classified as two main classes: (1) inpostmitotic cells: neuron degeneration (Alzheimer's, Parkinson's,stroke), sarcopenia (loss of muscle), cardiovascular diseases (heartfailure, myocardial infarction); and (2) in mitotic cells: loss of bonemarrow, degraded immune function, diabetes, idiopathic pulmonaryfibrosis, age-related macular degeneration, rheumatoid arthritis,osteoarthritis, osteoporosis, atherosclerosis, and hypertension. Morespecifically, Age-related diseases or disorders associated withmitochondrial dysfunction or/and telomere dysfunction include, but arenot limited to, cancer, osteoarthritis, age-related maculardegeneration, idiopathic pulmonary fibrosis, Parkinson's disease,Alzheimer's disease, Huntington's disease, skin aging, cataract,multiple sclerosis, Sjogren, Rheumatoid arthritis, atherosclerosis,myocardial infarction, heart failure, hypertension, stroke, diabetesmellitus, osteoporosis, obesity, grey hair, hearing loss, and so on. Allof the above-mentioned diseases or disorders are encompassed by thepresent invention.

In some preferred embodiments, the term “age-related disease ordisorder” refers to a disease or disorder selected from tumorigenesisand malignant cancer development, myocardial infarction (heart attack),cerebellar infarction, stroke, Parkinson's disease, heart failure,atherosclerosis, hypertension, cataract, age-related maculardegeneration, sarcopenia, osteoarthritis, osteoporosis, loss of bonemarrow, multiple sclerosis, Sjogren, Rheumatoid arthritis, degradedimmune function, diabetes, Idiopathic pulmonary fibrosis, andneurodegenerating disease, Alzheimer's disease, Huntington's disease,and disorders caused by the decline in testosterone, estrogen, growthhormone, IGF-I, or energy production.

As used herein, the term “anti-aging effect” refers to phenotypescomprising increased mitochondrial biogenesis and function, reduced ROSlevels, extended life span of senescent cells and post-mitotic cellssuch as neuron cells, prevented age-related disorders, such astumorigenesis, malignant progression of cancers, cerebellar infarctionand myocardial infarction.

As used herein, the term “prevent age-related disease or disorder” meansreducing the incidences, delaying or reversing the diseases related toaging.

As used herein, the term “senescence” refers to a cell cycle-arrestedstate in mitotic cells, which can be induced by telomere dysfunction,DNA damage, or oncogene activation. In budding yeast, senescent cellscaused by telomere dysfunction are arrested at the G2/M phase of thecell cycle. In mammalian cells, senescent cells are arrested at the G0phase, which is a non-dividing phase outside of the cell cycle.Senescence in WI-38 fibroblasts means that cells show no increase innumber under the microscope for 10 days after passage and exhibitβ-galactosidase positive staining.

As used herein, the term “post-mitotic cells” refers to a group of cellsthat are in arrested state at G0, which is a non-dividing phase outsideof the cell cycle, but continue to perform their main functions for therest of the organism's life. Post-mitotic cells include neuronal cells,heart muscle cells, and muscle cells. Some cell types in matureorganisms, such as parenchymal cells of the liver and kidney, enter theG0 phase semi-permanently and can only be induced to begin dividingagain under very specific circumstances. These types of cells can alsobe considered as post-mitotic cells when they are in G0 phase.

As used herein, the term “cdc13-1” refers to yeast mutant cells thatcontain a point mutation in the gene CDC13. The term “cdc13-1” alsorefers to the point mutant gene, while cdc13-1p refers to the proteinproduced by the point mutant gene cdc13-1, and Cdc13p refers to the wildtype protein.

As used herein, the terms “cdc13-2”, “stn-1”, “cdc17-1”, and “cdc17-2”refer to yeast mutant cells, or the corresponding mutant genes. Theterms “est1”, “est2”, “est3”, “hdf1”, and “hdf2” refer to yeast mutantcells containing a deletion of the gene EST1, EST2, EST3, HDF1 or HDF2,respectively, or refer to the corresponding gene deletions respectively.A person of ordinary skill in the art would readily understand the usageof these terms within the context.

As used herein, the term “TOR inhibitor” refers a class ofimmunosuppressive compounds which contain the basic rapamycin nucleus,including rapamycin and chemically or biologically modified derivativesthereof, which retain the ability to maintain senescence. Accordingly,the term “TOR inhibitor” may be in the form of ester, ether, hydrazone,hydroxylamine, or oxime derivatives of rapamycin. The term may alsoinclude analogs of rapamycin through modification of the functionalgroups on the rapamycin nucleus, for example, through reduction oroxidation reactions. Thus, the term “TOR inhibitor” includes, but is notlimited to rapamycin and analogs such as AP23573 (Deforolimus),AP-23675, AP-23841, ABT-578 (Zotarolimus), CCI779 (Temsirolimus),RAD-001 (Everolimus), 7-epi-rapamycin, 7-thiomethyl-rapamycin,7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin, and7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-rapamycin,and 42-O-(2-hydroxy)ethyl rapamycin.

As used herein, the term “therapeutic doses of rapamycin” means the doserange of rapamycin from about 1 mg/day to about 5 mg/day, which can beexpanded to from about 0.1 mg/day to about 15 mg/day, but does notexceed 40 mg/day in clinic. Under these doses, rapamycin inhibitsprotein translation and cell cycle progress at G1 phase, as well asinduces autophagy. In animal and tissue cultures, the therapeutic dosesmean above 0.1 mg/kg/day in marine models, above 10 ng/mL for humancells and above 100 ng/mL for marine cells, respectively. It isunderstandable to a person of ordinary skill in the art that the exacttherapeutic dose may vary for a specific cell line or animal.

As used herein, the term “low doses of rapamycin” means doses below “thetherapeutic doses”. For example, the low dose can be 0.1 to 1000 pM inserum medium for cells, 0.01 to 100 μg/kg/day in marine models, or 0.01to 100 μg/day in humans. The specific concentration is dependent on thespecific type of cells or diseases to be treated and the path ofadministration. These low doses can also be presented as about 0.001% toabout 10% of therapeutic doses of rapamycin.

As used herein, the term “anti-aging biological concentration” of acompound or composition means the concentration of an anti-agingbiologically active compound or composition, in contrast toconcentration of a compound or composition. The anti-aging biologicalactivity can be measured by senescence prolongation in cdc13-1 cells.

As used herein, the term “cancer” describes a diseased state in which anormal cell first becomes an abnormal cell with initial lesions such asDNA damages, oncogene activation, telomere dysfunction, and then becomesinvasive to adjacent tissues, to regional lymph nodes and to distantsites. Cancer can be age-related cancers, mutagen-induced cancers,secondary cancers induced by anticancer therapies or therapies againstan independent disorder or disease such as hormone replacement, orinduced by environments such as UV, ironing irradiation, and smoking.

As used herein, the term “preventing tumorigenesis” means to inhibit thetransformation of a normal cell into an abnormal cell, or to inhibit theformation of benign tumors. The term “preventing malignant progression”means to inhibit the development of benign tumors into malignant tumorsor cancers. The term “preventing cancer” or “cancer prevention” means toprevent tumorigenesis and/or to inhibit malignant progression.

As used herein, the term “cancer chemopreventive agent” refers a naturalor laboratory-made substance that can be used to inhibit tumor growth.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium generally accepted in the art for the delivery ofbiologically active agents to animals, in particular, mammals,including, e.g., adjuvant, excipient or vehicle, such as diluents,preserving agents, fillers, flow regulating agents, disintegratingagents, wetting agents, emulsifying agents, suspending agents,sweetening agents, flavoring agents, perfuming agents, antibacterialagents, antifungal agents, lubricating agents, and dispensing agents,depending on the nature of the mode of administration and dosage forms.Pharmaceutically acceptable carriers include both aqueous andnon-aqueous liquid media, as well as a variety of solid and semi-soliddosage forms. Such carriers can include a number of differentingredients and additives in addition to the active agent, suchadditional ingredients being included in the formulation for a varietyof reasons, e.g., stabilization of the active agent, binders, etc., aswell known to those of ordinary skill in the art.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt or zwitterionic form of a compound, which is water or oil-solubleor dispersible and, within the scope of sound medical judgment, suitablefor use in contact with the tissues of a patient without excessivetoxicity, irritation, allergic response, or other problem orcomplication commensurate with a reasonable benefit/risk ratio, and iseffective for its intended use. Representative acid addition saltsinclude acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorsulfonate; hemisulfate,heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide,hydroiodide, lactate, maleate, mesitylenesulfonate, methanesulfonate,naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,palmate, pectinate, persulfate, pivalate, propionate, succinate,tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate,bicarbonate, p-toluenesulfonate. Examples of acids which can be employedto form pharmaceutically acceptable addition salts include inorganicacids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, andorganic acids such as oxalic, maleic, succinic, and citric.

The cations of pharmaceutically acceptable salts include lithium,sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxicquaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, tetrabutylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine,pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine. Other representative organic amines useful for theformation of base addition salts include ethylenediamine, ethanolamine,diethanolamine, piperidine, and piperazine.

As used herein, the term “solvate” means a physical association of acompound identified according to this invention with one or more,preferably one to three, solvent molecules, whether organic orinorganic. This physical association includes hydrogen bonding. Incertain instances the solvate will be capable of isolation, for examplewhen one or more, preferably one to three, solvent molecules areincorporated in the crystal lattice of the crystalline solid. Thesolvent molecules in the solvate may be present in a regular arrangementand/or a non-ordered arrangement. The solvate may comprise either astoichiometric or nonstoichiometric amount of the solvent molecules.“Solvate” encompasses both solution-phase and isolable solvates.Exemplary solvates include, but are not limited to, hydrates,ethanolates, methanolates, and isopropanolates. Methods of solvation aregenerally known in the art.

In addition, compounds encompassed by the present invention may haveprodrug forms. Any compound that will be converted in vivo to providethe bioactive agent is a prodrug within the scope of the invention.Various forms of prodrugs are well known in the art. For examples ofsuch prodrug derivatives, see Design of Prodrugs, edited by H. Bundgaard(Elsevier, 1985), and Methods in Enzymology, Vol. 112, at pp. 309-396,edited by K. Widder et al. (Academic Press, 1985); and A Textbook ofDrug Design and Development, edited by Krosgaard-Larsen and H.Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H.Bundgaard, at pp. 113-191 (1991).

As an illustrative example, compounds containing a hydroxyl group, suchas rapamycin and its analogs, can form physiologically hydrolysableesters, carbonates, or carbomates that serve as prodrugs by beinghydrolyzed in the body to yield the parent compounds. Thus, the presentinvention encompasses use of rapamycin and analogs or theircorresponding ester, carbonate, or carbomate derivatives as anti-agingagents. These prodrugs can be synthesized by a person of ordinary skillin the art by using conventional synthetic methods known in the art.Just to illustrate, the esters include, but are not limited to, thosederived from acylation of the hydroxyl group(s) with an acylating agentknown to a person of ordinary skill in the art, for example, aceticanhydride, acetyl chloride, acetic acid, propionyl chloride, benzoylchloride, butyryl chloride, succinic anhydride, and so on. Thecarbonates include, but are not limited to, those derived from reactionof the hydroxyl group(s) with a compound having a structure of formulaX—C(O)OR, wherein X is a halide and R can be any group having a carbonattached to the oxygen atom, e.g., alkyl, aryl, arylalkyl, etc.Carbomates can also be synthesized in a similar manner.

In addition, prodrugs of rapamycin can also be in other forms asdescribed in U.S. Pat. No. 4,650,803 and U.S. Pat. No. 5,151,413, or inany other literature published or yet to be published, which are hereinincorporated by reference in their entirety. Most prodrugs describedherein are preferably administered orally since hydrolysis in manyinstances occurs principally under the influence of the digestiveenzymes. Parenteral administration may be used where a prodrug itself isactive, or in those instances where hydrolysis occurs in the blood.

Formulations of the present invention may be administered in anysuitable route known in the art, for example, by oral, topical,parenteral (including subcutaneous, intramuscular, intravenous andintradermal), and pulmonary route. In some embodiments, formulations areconveniently presented in unit dosage form and are prepared by anymethod known in the art of pharmacy. In general, the formulations areprepared by uniformly and intimately bringing into association of theactive ingredient (e.g., rapamycin or analogs thereof) with liquidcarriers or finely divided solid carriers or both.

Oral formulations containing the active compounds of this invention maycomprise any conventionally used oral forms, including tablets,capsules, troches, buccal forms, lozenges and oral liquids, suspensions,or solutions, or as a powder or granules, a solution or suspension in anaqueous or non-aqueous liquid, an oil-in-water liquid emulsion, or awater-in-oil liquid emulsion. Capsules may contain mixtures of theactive compound(s) with inert fillers and/or diluents. Useful tabletformulations may be made by conventional compression, wet or drygranulation methods, and may utilize pharmaceutically acceptablediluents, binding agents, disintegrants, lubricants, surface modifyingagents (including surfactants), or suspending or stabilizing agents.Oral formulations may utilize standard delay or time releaseformulations to alter the absorption of the active compound(s).

In some cases it may be desirable to administer the compounds in theform of an aerosol directly to the airways, ears, skin, or throat.

Rapamycin at a low dose may also be administered topically. The topicalforms include, but are not limited to, creams, ointments, emulsions,gels, lotions, and sprays. In one embodiment of the topical formulationsof the invention, the topical formulation comprises inert materials(such as oil). In one embodiment of the topical formulations of theinvention, the ingredients of the topical formulation are provided in amoisturizing cream base. Preservatives may also be provided in thetopical formulations of the invention to increase the formulation'sshelf life. Those skilled in the art would know how to modify thetopical formulations of the invention by adding additional activeingredients or inert materials. The topical formulations of theinvention may be used to prevent skin aging and to treat early stages ofdisorders such as cancers.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more pharmaceutically acceptable carriers made bycompressing or molding the respective agents. In preferred embodiments,compressed tablets are prepared by compressing in a suitable machine theactive ingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent.

The compounds or compositions of the present invention can beadministered alone or in combination with one or more, preferably one tothree, additional therapeutic agents. By “administered in combination”or “combination therapy” it is meant that the compound or composition ofthe present invention and one or more, preferably one to three,additional therapeutic agents are administered concurrently to themammal being treated. When administered in combination, each componentmay be administered at the same time or sequentially in any order atdifferent points in time. Thus, each component may be administeredseparately but sufficiently closely in time so as to provide the desiredtherapeutic effect.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that throughout the application data isprovided in a number of different formats and that these data representendpoints and starting points and ranges for any combination of the datapoints. For example, if a particular data point “2%” and a particulardata point “4%” are disclosed, it is understood that greater than,greater than or equal to, less than, less than or equal to, and equal to2% and 4% are considered disclosed as well as between 2% and 4%. It isalso understood that each unit between two particular units are alsodisclosed. When the term “about” appears in front of a number denotingdosage, it means that the value can vary by at least ±30%, preferably bywithin ±20%, and more preferably by within ±10%; when the term appearsin front of a number denoting temperature, it means that the value canvary by at least ±2° C., and more preferably by within ±1° C.; whendenoting time, it means that the value can vary by at least 15%,preferably by within 10%, and more preferably by within 5%.

Methods of Identifying, Detecting and Purifying Anti-Aging Agents

This invention is based on various discoveries discussed below.

A. Inhibition of Nutrient Signals Prolongs the Cell Cycle-Arrested StateInduced by Telomere Dysfunction in Yeast Via the AMPK and SubsequentMitochondrial Pathway.

A-1. Inhibition of Nutrient Signals Maintains the Cell Cycle-ArrestedState and Thus Prevents Subsequent Cell Death Induced by TelomereDysfunction.

The budding yeast cdc13-1 is an important model of telomere dysfunctionbased on the various reasons. For example, first, the telomeredysfunction induced by inactivation of cdc13-1p leads to the samedownstream pathway as that by inactivation of telomerase: Mec1 (ATM andATR homologue)-dependent cell cycle arrest at G2/M followed by massivecell death, which is accompanied by enlarged cell size, dramaticallyincreased ROS production, apoptotic markers, and more than 2N DNAcontent in haploid cells. Inhibition of TOR by rapamycin prevents thecell death induced by inactivation of cdc13-1p or by inactivation oftelomerase. Interestingly, rapamycin does not affect the G2/M arresttriggered by inactivation of cdc13-1p, but maintains the G2/M-arrestedstate and thus prevents the appearance of >2N DNA content, an indicatorof deterioration of senescence and cell death (Qi, H., et al., PLoS ONE,3, e3520 (2008)). Second, telomere structure and function are conservedfrom yeast to human. In human cells, telomere dysfunction also leads toATM-/ATR-dependent cell cycle arrest followed by massive cell deathmanifested by enlarged cell size, apoptotic markers and polyploidy(Denchi, E. L., et al, Nature, 448:1068-71 (2007); Shay, J. W., et al,Carcinogenesis, 26:867-74 (2005)), similar to that in cdc13-1. Thus,cdc13-1 can be used to study telomere dysfunction-induced downstreamcascade.

This invention discloses that, similar to a rapamycin treatment, theglucose restriction by reducing glucose in the yeast culture medium orby addition of 200 μM 2-deoxyglucose (an analogue of glucose) to themedium, and the nitrogen limitation also prevented cell death induced byinactivation of cdc13-1p as monitored by the colony formation assay. Asshown in FIG. 1A, incubation of cdc13-1 cells (haploid) at thenon-permissive temperature (37° C.) for 22 hrs in regular YEPD medium(1% peptone, 2% yeast extract, and 2% glucose) resulted in massive lossof viable cells as measured by the colony formation assay. However,reducing glucose in YEPD medium (from 2% to 1%, 0.5% and 0%) andaddition of 200 μM 2-deoxy-glucose to YEPD with 2% glucose preventedcell death effectively: less glucose, more survived cells. In addition,limitation of nitrogen by SC-N medium (containing 0.67% yeast nitrogenbase in absence of amino acids and (NH₄)₂SO₄, 2% glucose, plus a mixtureof 100 mg/L each of histidine, leucine, tryptophan and uracil for thisyeast strain) prevented the cell death (FIG. 1B). Furthermore, thecdc13-1 cell death was prevented by rapamycin in a dose-dependent mannerwhen the concentrations were between 0.3 to 1 nM (FIG. 1C). 0.5 and 1 nMof rapamycin that prevented the cell death did not inhibit, but slightlypromoted, cell growth consistently (FIG. 1D), indicating a novelfunction of rapamycin at a low dose. FIG. 1E shows that rapamycin andglucose restriction delay cdc13-1 cell death. In contrast to dramaticcell loss after 20 hrs at 37° C., low doses of rapamycin and glucoserestriction resulted in a slow-loss of cells. After 60 hrs of incubationat 37° C., cell survival was at least 100-fold more than those withouttreatment.

The results further demonstrate that glucose restriction, similar torapamycin treatment, maintains the G2/M-arrested state and preventssubsequent cell death induced by inactivation of cdc13-1p. As shown inFIG. 2A, after incubating cdc13-1 cells at the non-permissivetemperature (37° C.) for 2 hrs, more than 95% of cells entered the G2/Mphase of cell cycle in glucose restriction (0.5% glucose) and rapamycin(1 nM) containing YEPD medium, similar to that in the control. Glucosereduction from 2% to 0.5% in YEPD medium inhibited the increase of >2NDNA content, suggesting maintenance of the cell cycle-arrested state,same as the rapamycin treatment (1 nM). As shown in FIG. 2B,pre-incubation of cdc13-1 cells at 37° C. for two hrs, which allowsgreater than 95% of cells to enter the G2/M phase of cell cycle, did notaffect the preventive effect of glucose restriction and rapamycin on thecell survival. Thus, nutrient limitation, similar to TOR inhibition byrapamycin, maintains the cell cycle-arrested state (the senescentstate), and thus prevents the deterioration of senescence and theresulting cell death upon telomere dysfunction.

A-2. Glucose Restriction, Same as TOR Inhibition by Rapamycin, Preventsthe Induction of ROS and Inhibits the Appearance of Apoptotic Markers incdc13-1 Cell Death.

FIG. 3A shows that cdc13-1 cell death is associated with a dramaticincrease in ROS release. Similar to rapamycin (1 nM) treatment,reduction of glucose from 2% to 0.5% (0.5% Glc), effectively reduced ROSincrease to a level comparable to that of wild type (WT) at the sametemperature. FIG. 3B shows that cell death induced by inactivation ofcdc13-1p is also associated with increased apoptosis as monitored byphosphatidylserine (PS) flipping and glucose reduction (0.5% Glc) orrapamycin (1 nM) effectively inhibits this apoptotic death.

A-3. AMPK and Mitochondrial Function Play Important Roles in thePreventive Effects of Rapamycin and Glucose Restriction on the cdc13-1Cell Death.

This invention also discloses the important role of AMPK for thepreventive effects on the cdc13-1 cell death. FIG. 4A shows thatdeletion of Sip2p, the regulatory beta subunit of yeast AMPK,significantly inhibited the prevention effect of glucose restriction.Furthermore, deletion of Snf1p and Snf4p, the catalytic alpha subunitand the regulatory gamma subunit respectively, greatly reduced thepreventive effect of rapamycin (FIG. 4B).

This invention further discloses that mitochondria, whose function canbe improved by AMPK activation and the downstream mitochondrialbiogenesis, play an important role in the preventive effects on thecdc13-1 cell death. The mitochondrial deficient mutation was made incdc13-1 cells by incubating cells in ethidium bromide containing mediumas described in a previous published paper (Qi, H., J. Biol. Chem.,278:15136-15141 (2003)). FIG. 5A shows that the mitochondria deficiencysignificantly abolished the preventive effect by glucose reduction andrapamycin (at 1 nM and 5 nM). Up-regulation of mitochondrial biogenesisby glucose reduction (0.5% glucose) and rapamycin (1 nM) was alsodemonstrated by measuring the increase of mitochondrial mass (FIG. 5Band FIG. 5C).

FIG. 6 shows the summary of above results. Mitochondria play animportant role in maintaining the growth-arrested state induced bytelomere dysfunction, and glucose restriction and TOR inhibition byrapamycin stimulate mitochondrial function through AMPK activation, thusprevent deterioration of senescence and subsequent cell death.

B. Low Doses of Rapamycin, Glucose Restriction, and an AMPK Activator,in a Similar Manner, Stimulate Mitochondrial Function and ProlongSenescence in Primary Human Fibroblasts.

This invention discloses that CR or rapamycin also prolongs senescenceand prevents cell death induced by telomere dysfunction in human cells.Primary human embryonic lung fibroblasts WI-38 were used. These cellslack telomerase activity and exhibit telomere dysfunction upon reachingreplication potential, usually at passage 31 under routine cultureconditions. Cells at passage 29 were treated with 50 pM rapamycin. Thetreatment was on a 10-day cycle of 3-day with and 7-day withoutrapamycin (also termed as 3-day-on/7-day-off cycle). Cells ceaseddividing at passage 31 (examined under a microscope) and exhibitedsenescence in the presence or absence of rapamycin by measuring thecytosol β-galactosidase activity using 5-Bromo-4-chloro-3-indolylβ-D-galactoside (X-gal) in buffer containing 1 mg/mL X-gal, 40 mM citricacid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mMpotassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂. Due to increasedcytosol β-galactosidase activity, senescent cells are stained a bluecolor (shown as dark grey in black/white picture). FIG. 7 shows thatafter 56-days in senescence, massive cell loss occurred in the DMSOcontrol. Cell loss was strongly inhibited by 50 pM rapamycin. Rapamycindidn't transform cells since the senescence marker was still present andcell growth was not observed. In addition, reduced glucose in theculture medium (from 0.4% to 0.2%) or addition of 2-deoxyglucose (12.5μM) also prevented loss of senescent cells (FIG. 7).

Similar to the observation in yeast, AMPK also plays an important rolein rapamycin- or glucose restriction-mediated prevention oftelomere-death in human cells. As shown in FIG. 7, treatment with AMPKspecific activator5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR, 250 μM)(in a 10-day cycle of 2-day with and 8-day without AICAR to avoidtoxicity) prevented the loss of senescent WI-38 cells. In addition,Rapamycin (50 pM) activated AMPK kinase activity as monitored by Westernblotting, which was used to detect the phosphorylated AMPK at Thr172(data not shown). Furthermore, rapamycin, reduced glucose,2-deoxyglucose, and AICAR increased mitochondrial mass and stimulatedmitochondrial function in humans (FIG. 8 and FIG. 10). Low doses ofrapamycin increased mitochondrial mass in human fibroblasts (FIG. 8A) aswell as in lymphoblastoid L40 cells (data not shown). Rapamycin at lowdoses also increased mitochondrial membrane potential and reduced ROS infibroblasts and in L40 cells (FIG. 8B, FIG. 8C, and data not shown).

Surprisingly, only low doses of rapamycin (from 10 to 100 pM) preventedthe loss of senescent WI-38 cells as shown in FIG. 9A. Rather, rapamycinat a concentration higher than 500 pM promoted cell loss. For example,25 pM rapamycin prevented cell loss (FIG. 9A), but did not inhibit cellgrowth (FIG. 9B). Instead, it increased the replication potential asmeasured by population doubling from 5.12 to 6.8. In addition, rapamycinat low doses (50 pM and 100 pM) increased the levels of the keysenescence proteins p53, p21 and pRB, but not at a high dose (2000 pM)(FIG. 9C). Furthermore, only low doses of rapamycin increasedmitochondrial mass and membrane potential and reduced ROS levels inhuman cells, whereas an amount greater than 10 nM of rapamycin appearsto lose this effect (FIG. 8). Thus, rapamycin at low doses functionsdifferently from that of therapeutic doses, it stimulates mitochondrialfunction and prevents deterioration of senescence, rather thaninhibiting protein translation and cell growth at cell cycle G1 phase.

In summary, 1) the maintenance of the cell cycle-arrested state insenescent cells is conserved from yeast to human; 2) CR, glucoserestriction or a low dose of rapamycin stimulates mitochondrial functionvia AMPK, prolongs senescence and thus inhibits the subsequent celldeath; and 3) only low doses of rapamycin mimic caloric restriction interms of maintaining the senescent state in human cells.

C. Several Anti-Aging and Cancer Chemopreventive Agents StimulateMitochondrial Function and Inhibit Loss of Senescent Cells in Both Yeastand Human Cells.

This invention discloses that two agents known to prevent a number ofage-related diseases, EGCG from Green tea extract (GTE) and Grape seedextract (GSE), can increase mitochondrial mass and prolong senescence.

GTE has been reported to prevent various cancers (U.S. Pat. Nos.7,192,612 and 7,384,655, and US Application Publication Nos. 20040142048and 20040047921, and Shimizu, M., et al., Cancer Epidemiol. BiomarkersPrey., 17:3020-3025 (2008); Nakachi, K., et al., Jpn. J. Cancer Res.,89:254-261 (1998)). Epigallocatechin gallate (EGCG) is the mosteffective components of GTE. EGCG has been reported to extend life spanin C. elegans (Abbas, S. and Wink, M., Planta. Med., 75:216-221 (2009)).It has also been reported to modulates amyloid precursor proteincleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice(Rezai-Zadeh, K., et al., J. Neurosci., 25(38):8807-8814 (2005)),prevent brain damage after transient middle cerebral artery occlusion inrats (Choi, Y. B., et al, Brain Res., 1019:47-54 (2003)), protectcardiac myocytes from ischemia/reperfusion-induced apoptosis and limitinfarction size via mitochondrial K(ATP) channel activation in isolatedrat hearts (Townsend, P. A., et al., FASEB J., 18:1621-1623 (2004);Song, D. K., et al, J. Korean Med. Sci., 25(3):380-386 (2010)).Furthermore, it has been demonstrated to prevent apoptosis in isolatedislets (Hara, Y., et al., J. Hepatobiliary Pancreat. Surg., 14:493-497(2007)), prevent autoimmune diabetes induced by multiple low doses ofstreptozotocin in mice (Song, E. K, et al, Arch. Pharm. Res., 26:559-563(2003)), reduce autoimmune symptoms in a murine model of human Sjogren'ssyndrome (Hsu, S. D., et al., Autoimmunity, 40:138-147 (2007)), andprevent cataractogenesis induced by selenite in a rat model (Gupta, S.K., et al., Ophthalmic Res., 34:258-263 (2002)). Interestingly, EGCGalso activates AMPK (Huang, C. H., et al., Mol. Nutr. Food Res.,53(9):1156-1165 (2009)).

GSE has also been shown to have cancer prevention activities (US PatentApplication Publication Nos. 20040047921 and 20050013880). For example,gallic acid, a major constituent of GSE, reduces progression of prostatecancer to advanced stages in the TRAMP mouse model (Raina, K., et al.,Cancer Res., 67:5976-5982 (2007)) and GSE also prevents thecarcinogenesis of precancerous human breast epithelial cell MCF10Ainduced by NNK (Siriwardhana, N, et al., Breast Cancer Res. Treat.,109:427-441 (2008)). In addition, GSE has been shown to reduce arterialpressure and salt-sensitive hypertension in estrogen depleted animalmodels (see review by Carlson, S., et al., Gend. Med., 5 Suppl. A,S76-90 (2008)). It can also effectively prevent UVB-induced skin damagein a three-dimensional tissue culture model of human epidermis (Tomaino,A., et al., Toxicol. In Vitro., 20:1395-1402 (2006)), prevent Aβoligomerization and attenuate cognitive deterioration in a mouse modelof Alzheimer's disease (Wang, J., et al, J. Neurosci., 28:6388-6392(2008)), prevent high-fat diet-induced obesity in mice and hamsters(Park, S. H., et al, Nutr. Res. Pract., 2:227-233 (2008); Décordé, K.,et al, Mol. Nutr. Food Res., 53:659-666 (2009)), inhibit theaccumulation of oxidatively damaged DNA due to aging in the spinal cordand brain in a rat model (Balu, M., et al., Brain Res. Bull., 68:469-473(2006)), and maintain the integrity of the erythrocyte membrane duringaging (Sangeetha, P., et al., Exp. Gerontol., 40:820-828 (2005)).

As shown in FIG. 10, both 250 μg/mL GSE and 50 μg/mL EGCG from GTEincreased mitochondrial mass in human cells, for example, inlymphoblastoid L40 cells. They also prevented telomere-death in yeastand human primary fibroblasts WI-38 (FIG. 11 and FIG. 7)

FIG. 10 shows that a number of cancer chemopreventive agents increasemitochondrial mass in human lymphoblastoid cells, for example, 10 μMLY294002 (a PI3K inhibitor), 2 μM diallyl trisulfide (DATS), 1 μM benzylisothiocyanate (BITC), 1 μM phenyl isothiocyanate (PITC), 2 μg/mLresveratrol (RSV) and 0.03 μM lycopene, 6.7 μM phenethyl isothiocyanate(PEITC), 1 μM allyl isothiocyanate, 5 mM silibinin, 1.25 mM selenite(Na₂SeO₃), 2.5 mM genistein, and 3 mg/mL bilberry extract. FIGS. 7 and11 show that a number of cancer chemopreventive agents, includingphenethyl isothiocyanate (PEITC), silibinin, selenite (Na₂SeO₃),genistein, and bilberry extract, exhibited various degrees of theprotective effects on telomere-death in yeast and/or in human cells.

D. A number of Age-Related Diseases or Disorders are Associated withDysfunction of Mitochondria or/and Telomeres, and Low Doses of RapamycinPrevent Several Age-Related Diseases or Disorders in Animal Models orTissue Culture Models.

Cancers. Almost all cancer cells exhibit compromised mitochondrialfunction. This phenomenon is termed as Warburg effect, which means thatas much as 60% of ATP is produced through glycolysis under aerobicconditions in cancer cells, whereas in normal cells, most of the ATP isgenerated through the mitochondrial oxidative phosphorylation. Oncogenictransformation has been shown to result in suppression of oxidativephosphorylation and an increase in glycolysis, whereas the tumorsuppression protein p53 upregulates respiration and suppressesglycolysis. However, it is not clear whether compromised mitochondrialfunction is the cause or the result of cancers.

It is known that telomere dysfunction is age-dependent due toprogressively shortening of telomeres, and maintaining senescenceinduced by telomere dysfunction is a key mechanism for preventingage-related cancer development. As senescence induced by oncogeneactivation and mutagens is also via DNA damage response, same as that bytelomere dysfunction, senescence maintenance may be an intrinsicmechanism for preventing various cancers.

This invention discloses that mitochondrial function plays a key role inmaintaining senescence. Thus, mitochondrial function is important incancer prevention via maintaining senescence and compromisedmitochondrial function is an early step in promoting senescencedeterioration and cancer development. Therefore, the telomeredysfunction model can be used to identify candidates for stimulatingmitochondrial function, prolonging senescence and preventing cancers.Indeed, a number of known cancer chemoprevention agents were shown toprolong senescence and increase mitochondrial mass (FIGS. 7, 10 and 11).Furthermore, the low dose of rapamycin and AICAR, which prolongedsenescence as shown in FIG. 7, reversed the decrease in mitochondrialmass and prevented tumorigenesis induced by mutagen TPA(12-O-tetradecanoylphorbol 13-acetate) (FIG. 12 and FIG. 13) in NIH3T3cells (see Example 15).

Neuron Degenerative Diseases. In spite of extensive studies, themechanisms of neuron degenerative diseases are not clear. Mitochondrialdysfunction may play a role in the diseases, since Pakin (linked toParkinson's disease), Huntintin (linked to Huntington's disease andamyloid-β(causes senile plaques in Alzheimer's disease) are involved inmitochondrial function. Recent studies also suggest that autophagy(protein degradation) plays a role in these diseases.

This invention discloses that the low doses of rapamycin, but not higherdoses of rapamycin, reduce ROS levels, increase the life span of ratcerebellar granule neuron (CGN) cells in culture (FIG. 14A and FIG. 14B)and prevent brain damage due to cerebellar infarction in a rat strokemodel (FIG. 15A and FIG. 15B) (see Examples 16-18). Rapamycin at lowdoses also reduces ROS levels induced by 200 μM MPP⁺, the dorpaneurictoxin used to induce Parkinson's in marine models (FIG. 16).Furthermore, EGCG, which has been reported to prevent or treat neurondegenerative diseases, can also prolong senescence (FIG. 7 and FIG. 11).Thus, the low doses of rapamycin and EGCG can prolong the life span ofboth post-mitotic and senescent cells, suggesting that maintenance ofthe G0 phased post-mitotic neuron cells share similar mechanisms tothose of the G0 phased senescent cells. Therefore, in one aspect, thepresent invention is based on the discovery that the senescent model canbe used to identify and detect drug candidates that prevent neurondegenerative diseases or disorders, including but not limited to stroke,Parkinson's, Alzheimer's, and Huntington's diseases.

Heart Failure, Atherosclerosis and Myocardial Infarction. Telomeredysfunction has been shown to play an important role in chronic heartfailure. It has been shown that in cultured cardiomyocytes, interferencewith TRF2 function triggered telomere erosion and apoptosis. Conversely,exogenous TRF2 conferred protection from oxidative stress, indicatingthat cell death can occur via telomere dysfunction even in post-mitotic,noncycling cells (Oh, H., et al., Proc. Natl. Acad. Sci. USA,100:5378-5383 (2003)). In vivo, the aged 5th-generation TERC-deficientmice (G5TERC-KO) (a telomerase mutant model due to deficiency intelomerase RNA encoded by TERC gene) exhibited significantly shortertelomeres in cardiomyocytes, ventricular dilation, thinning of themyocardium, cardiac dysfunction, and sudden death. Heart sections fromthe G5TERC-KO mice revealed an increased expression of DNA damageresponse protein p53 and increased apoptosis, as well as a 50% reductionin the number of left ventricular myocytes as compared with wild-typemice (Leri, A., et al., EMBO J., 22:131-139 (2003)).

Atherosclerosis is commonly referred to as a hardening or furring of thearteries. It is caused by the formation of multiple plaques within thearterie. The dysfunction of vascular endothelial cells (ECs) triggeredby atherogenic stimuli is of central importance in the pathogenesis ofatherosclerosis. Accelerated telomere erosion and premature senescencehave been observed in human atherosclerotic lesions (Ogami, M., et al.,Arterioscler. Thromb. Vasc. Biol., 24:546-550 (2004); Minamino, T., etal., Circulation, 105:1541-1544 (2002)), suggesting that dysfunction orloss of EC due to age-dependent telomere erosion may be an early step ofplaque formation.

Coronary atherosclerosis leads to the blockage of coronary circulationthat causes myocardial infarction (MI, commonly known as heart attack).MI leads to the loss of the post-mitotic cardiomyocytes, maladaptiveremodeling, cardiac contractile dysfunction and eventually congestiveheart failure.

This invention discloses that the low dose of rapamycin at 10 μg/kg, butnot at 100 μg/kg, significantly reduced the myocardial ischemicinfarction in a rat model (FIG. 17 and Example 19). Furthermore, it hasbeen reported that AMPK activator metformin also can reduce myocardialinfarction in marine models and protect myocardial cells frommitochondria-mediated cell death (Calvert, J. W., Diabetes, 57:696-705(2008)). These results demonstrate the importance of theTOR/AMPK/mitochondria pathway in maintaining the post-mitotic myocardialcells, similar to that in senescent cells. Thus, the senescence modelinduced by telomere dysfunction can be used to select drug candidatesfor myocardial infarction prevention or treatment.

Age-related Macular Degeneration. Age-related macular degeneration (AMD)is a major cause of blindness in the elderly (>50 years). It isinitiated from degeneration of retinal pigment epithelial (RPE) cellsand results in a loss of vision in the center of the visual field (themacula). It has been reported that telomere erosion, mitochondrialfunction loss and cell loss are associated with this disease (Matsunaga,H., Invest. Ophthalmol. Vis. Sci., 40:197-202 (1999); Liang, F. Q., etal., Exp. Eye Res., 76:397-403 (2003)). Therefore, improvingmitochondrial function to prevent cell loss induced by telomere erosioncan prevent or stabilize the early stages of disease. Interestingly, alow dose of rapamycin at 50 pM has been patented for treating thisdisease (U.S. Pat. No. 7,083,802). Thus, the senescence model induced bytelomere dysfunction can be used to select drug candidates forpreventing or treating AMD.

Osteoarthritis. Osteoarthritis (OA), a progressive loss of articularcartilage, is the most common chronic joint disease in the elderlypopulation, which causes significant pain and disability. The functionof the chondrocytes is essential to the maintenance of a propercartilage matrix. It has been shown that telomere dysfunction,mitochondrial mutations and apoptotic cell death in chondrocytes areassociated with OA (Martin, J. A., et al., J. Bone Joint Surg. Am., 85-ASuppl. 2:106-110 (2003); Ruiz-Romero, C., et al., Mol. Cell Proteomics.,8:172-189 (2009); Dave, M., et al., Arthritis Rheum., 58:2786-2797(2008)), suggesting that the telomere dysfunction-induced cell loss inchondrocytes is an underlying mechanism for the disease initiation.Therefore, improving mitochondrial function to prevent cell loss inducedby telomere dysfunction may prevent or stabilize the early stages of thedisease. Thus, the senescence model induced by telomere dysfunction canbe used to select drug candidates for preventing or treating OA.

Idiopathic Pulmonary Fibrosis. Idiopathic pulmonary fibrosis (IPF) is achronic, progressive interstitial lung disease, characterized as anabnormal and excessive deposition of fibrotic tissue in the pulmonaryinterstitium. It usually occurs in patients of greater than 50 years ofage. Recently, it has been shown that mutations in telomerase can leadto adult-onset pulmonary fibrosis (Tsakiri, K. D., et al., Proc. Natl.Acad. Sci. USA, 104:7552-7557 (2007)) and short telomeres are associatedwith IPF (Alder, J. K., et al., Proc. Natl. Acad. Sci. USA,105:13051-13056 (2008); Armanios, M. Y., et al., N. Engl. J. Med.,356:1317-1326 (2007)). The involvement of mitochondria and apoptosis inlung epithelial cells has also been demonstrated in IPF (Kuwano, P.,Intern. Med., 47:345-353 (2008)). These results suggest that telomeredysfunction-induced loss of lung epithelial cells may be an initialtrigger of the age-related IPF. Therefore, improving mitochondrialfunction for preventing cell loss induced by telomere erosion in lungepithelial cells may prevent or stabilize the early stages of disease.Thus, the senescence model induced by telomere dysfunction can be usedto select drug candidates for preventing or treating AMD.

Skin Aging. Fibroblast aging plays an important role in signs of skinaging such as wrinkles. Progressive shortening of telomeres andaccumulation of DNA damages by ROS or UV lead to fibroblast aging,including fibroblast senescence and subsequent cell loss. Fibroblastaging then results in functional loss, including loss of proliferativepotential (Mine, S., et al., PLoS ONE, 3(12):e4066 (2008); Hayflick, L.,J. Invest. Dermatol., 73:8-14 (1979)), changes in cell morphology andmetabolism, decline in the production of extracellular matrix proteinssuch as type I and III collagens (Varani, J., et al., Am. J. Pathol.,168:1861-1868 (2006)), and overexpression of proteases involved in thedegradation of the extracellular matrix (West, M. D., et al., Exp. CellRes., 184:138-147 (1989)). These changes in vitro may all more or lessparticipate in the in vivo age-related changes of the skin. Maintainingfunctional mitochondrial population not only slows telomere shortening(FIG. 9B), but also prevents loss of the senescent fibroblasts that arestill functional (FIG. 7 and FIG. 9A), and consequently, delays skinaging and prevents skin cancers.

Rheumatoid arthritis (RA). Dysfunctional telomeres and mitochondrialmutations are potential pathogenic factor in RA. It has been shown thathematopoietic precursor cells (HPCs) and bone marrow mesenchymal stemcells (MSCs) from RA patients display premature telomere shortening andreduced replication potential (Colmegna, I., et al., Arthritis Rheum.,58:990-1000 (2008); Kastrinaki, M. C., et al., Ann. Rheum. Dis.,67:741-749 (2008)). Furthermore, HLA-DRB1*04 alleles, the majorsusceptible genes for this disease, has been shown to regulate theprocess of telomere shortening (Schonland, S. O. , et al., Proc. Natl.Acad. Sci. USA, 100:13471-13476 (2003)). Several studies have also shownthat some characteristic changes in the composition and structure of theinflamed synovial membrane in RA are linked to an altered apoptoticresponse of synovial cells (Korb, A., et al., Apoptosis, 14:447-454(2009)). Furthermore, mutations of mtDNA of synoviocyte from RA aregreatly increased as compared to controls (Da Sylva, T. R., et al.,Arthritis Res. Ther., 7:R844-851 (2005)). Thus, drug candidates thatimprove mitochondrial function and prevent cell death induced bytelomere shortening may be developed to treat this disease.

Diabetes Mellitus. Diabetes mellitus refers to a group of diseases thatlead to high blood glucose levels due to a diminished production ofinsulin (in type 1) or resistance to its effects (in type 2 andgestational). Premature loss of β-cell function due to telomeredysfunction or mitochondrial dysfunction may be an initial step indiabetes mellitus. It is well accepted that mtDNA defects are a commonfactor in the etiology of diabetes, and mtDNA rearrangement (Ballinger,S. W., et al., Nat. Genet., 7:458-459 (2004); Ballinger, S. W., et al.,Nat. Genet., 1:11-15 (1992)) and tRNA mutations (van den Ouweland, J.M., et al., Diabetes, 43:746-751 (1994)) are linked to diabetes.Furthermore, inactivation of the mitochondrial transcription factorTFAM, one of the key proteins in mitochondrial biogenesis, in thepancreatic β-cells in mice, leads to progressive decline in β-cell massby apoptosis, resulting in a severe reduction in serum insulin andincreased blood glucose in both fasting and nonfasting states (Koster,J. C., et al., Cell, 100:645-654 (2000); Wallace, D. C., Am. J. Med.Genet., 106:71-93 (2001)). It has also been shown that β-cell in humanpancreatic islet undergoes telomere erosion and telomere-inducedsenescence in vitro (Halvorsen, T. L., J. Endocrinol., 166:103-109(2000)). Therefore, drug candidates that improve mitochondrial functionand prevent cell loss induced by telomere erosion may prevent thedisease.

The age-related diseases mentioned above are a few examples that areassociated with mitochondrial deterioration and/or telomere dysfunction.There are many other diseases that would fall into this category,including, but not limited to, obesity, osteoporosis, hypertension,sarcopenia, cataract, multiple sclerosis, Sjogren syndrome, age-relatedhearing loss, graying, age-related immune dysregulation, and disorderscaused by the decline in testosterone, estrogen, growth hormone, IGF-I,or energy production. Thus, the present invention also encompasses anyage-related diseases or disorders uncovered in the future.

In summary, this invention discloses that mitochondria play a vital rolein maintaining the growth-arrested state in senescent and post-mitoticcells such as neurons and cardiamyocytes and are associated with anumber of age-related diseases. In addition, mitochondria dysfunctionand telomere dysfunction have also been linked to age-related diseases.These principles are the basis to use the senescent cells to search fordrug candidates against age-related disease.

FIG. 18 summarizes the mechanism of age-related diseases and illustrateshow inhibition of the nutrient/TOR pathway prevents age-relateddiseases. In brief, mitochondria play a vital role in age-relateddiseases in various cells and tissues. In post-mitotic cells such asneuron cells, muscle cells and cardiomyocytes, mitochondria serve tomaintain the post-mitotic state, thus prevent cells from re-entering thecell cycle and the subsequent cell death. In proliferative tissues,improving mitochondrial function can result in a reduction of oxidativestress and increase their replication potential. Upon enteringsenescence, mitochondria also maintain the senescent state and preventsubsequent cell loss, as in post-mitotic cells. Cell loss could triggerrepairing cascades, such as inflammation response in RA and fibrosis inIPF disease. Cell loss could also lead to functional loss of the tissuesand degenerative diseases, such as bone marrow failure, neurondegeneration and heart failure. In another situation, senescencedeterioration could lead to tumorigenesis and cancer progression.Caloric restriction, including glucose restriction, and the low doses ofrapamycin stimulate mitochondrial function and prevent variousage-related disease or disorders.

In summary, this invention has disclosed, inter alia: (i) that senescentstate is maintained by mitochondrial function; (ii) maintenance of thepost-mitotic state is similar to that of senescence; (iii) CR throughmitochondrial function maintains senescent and post-mitotic cells, andthus senescence models can be used to identify candidates that stimulatemitochondrial function and prevent various age-related disorders; (iv) anumber of cancer chemoprevention agents, inhibitors of glucose intakeand AMPK activators are able to prolong senescence and thus can bedeveloped into drug candidates for age-related diseases; and (v) a lowdose of rapamycin, as a CR mimic, is able to maintain senescent and postmitotic cells and prevent cancer, brain damage in stroke, myocardialischemic infarction and other age-related diseases or phenotypes.

Very recently, after the provisional applications to which thisapplication claims priority were filed, several researchers reportedthat the therapeutic doses of rapamycin may also have some effect onaging and age-related diseases via protein translation inhibition anddegradation (autophagy) stimulation. For example, a therapeutic dose ofrapamycin (7.5 mg/kg) is reported to be effective for Parkinson'sdisease in a mice model via protein translation inhibition particularlyon RTP801/REDD1/Ddit4, a protein that is induced in affected neurons ofParkinson patients and causes neuron death (Malagelada, C., et al., J.Neurosci., 30(3):1166-1175 (2010)). Another report demonstrated that atherapeutic dose of rapamycin (2.24 mg/kg) rescues cognitive deficitsand ameliorates Aβ and tau pathology in a mouse model of Alzheimerdisease by increasing autophagy (Caccamo, A., et al., J. Biol. Chem.,Feb. 23, 2010). Furthermore, a modest effect on life-span extension(˜10%) in the mouse and fruit fly was shown to be induced by rapamycinat the therapeutic dose (Harrison, D. E., et al., Nature,460(7253):392-395 (2009)), as compared to the effect on life spanextended by CR (˜30-40%). Interestingly, such a life span extension byrapamycin in fruit fly is not via the AMPK/mitochondrial pathway, butvia protein translation inhibition and/or autophagy stimulation (Bejdov,I., et al., Cell Metab., 11(1):35-46 (2010)).

These results suggest that therapeutic doses of rapamycin could havesome modest effect on some age-related diseases and life span extensionvia protein translation/growth inhibition and autophagy stimulation.However, because of various adverse effects, the therapeutic doses ofrapamycin are not suitable for long-term use to prevent age-relateddiseases or disorders. On the other hand, low doses of rapamycin, as amimic of CR (in fact, better than CR due to its minimum adverse effect)as disclosed herein, can be used for preventing aging and age-relateddiseases via the AMPK/mitochondrial pathway, which has various apparentadvantages over the use of therapeutic doses of rapamycin discussedabove, for example, higher effectiveness and lesser adverse effects.

EXAMPLES

A. The Use of Telomere Dysfunction Model for High-Throughput Screeningto Identify and Detect Anti-Aging Candidates for Preventing or TreatingAge-Related Diseases or Disorders

In one aspect of the present invention, the yeast telomere dysfunctionmodel cdc13-1 is used for the high-throughput screening for discovery ofuseful anti-aging agents for the prevention or treatment of age-relateddiseases or disorders. The cdc13-1 model has been well studied and thecell cycle arrest occurs at the G2/M phase immediately after cdc13-1pinactivation, followed by cell death. By using this model, the presentinvention provides a rapid way to identify candidates that can preventtelomere dysfunction-induced cell death. Other models associated withtelomere dysfunction that exhibit this type of quick growth arrest andsubsequent cell death can also be used, for example, stn1-1, cdc17-1 andcdc17-2 temperature sensitive mutants in Stn1p (capping telomeres inyeast) and Cdc17p (the yeast catalytic subunit of the DNA polymerase Ialpha-primase complex) respectively. In addition, yeast cells thatexhibit telomere dysfunction and apoptosis can be used, which include,but are not limited to, mutations in Est1p, Est2p, Est3p, Hdf1p, Hdf2p,or cdc13-2 mutant. WI38 human primary fibroblasts then can be used asthe human telomere dysfunction model to confirm or detect drugcandidates. The followings (Examples 1-6) use the cdc13-1 model.

Example 1 Identification of Agents that Prevent Cell Death in cdc13-1Model Using Cell Surviving Assays (as Shown in FIG. 19)

For high-throughput screening of compounds or compositions that improvemitochondrial function, prolong the cell cycle arrested state andprevent cell death in cdc13-1 model, cdc13-1 cells are activated from−80° C. stock first by streaking them on a fresh YEPD or yeast completemedium (YC) plate and then incubating at the permissive temperature(about 24° C.) for 3 to 5 days until single colonies formed. A few yeastcolonies are picked and cultured overnight in the same liquid medium ata permissive temperature 24° C. The overnight cultures are diluted intofresh medium from 1:2 to 1:20 (optimized at about 1:10 for cdc13-1strain).

Yeast cells are transferred into 96-well plates or any suitable formatsof plates. Prospective compounds or compositions, for example, seriallydiluted drugs, compounds, peptide libraries or other libraries by H₂O,DMSO (dimethyl sulfoxide) or other organic solvents, are added into thecells with a volume less than 5% of total volume of the cell mixture. Inthe mixtures, the corresponding solvent is included as a negativecontrol, and a 1 nM rapamycin solution in the same solvent is includedas a positive control. Yeast cells are then incubated at anon-permissive temperature (about 37° C.) for two days, or until nosurviving cells in the negative control are detected.

A small volume of cells (e.g., 5 μl) is transferred to YEPD agar platespre-made in another 96-well or any other format of plates (Cells canalso be diluted first before transferring). Plates are incubated at apermissive temperature (about 24° C.) for at least 3 days or untilcolonies form in positive control. Chemicals that promote colonyformation will be considered as primary candidates. Alternatively, a fewmicro-liters (μl) of cells are transferred into YEPD liquid medium inanother 96-well plate or any suitable format of plates. The plates areincubated at a permissive temperature (about 24° C.), and OD₅₉₅ (opticaldensity at 595 nm) is read periodically, e.g., once a day. Chemicalsthat increase cell density more than the negative control are consideredto be primary candidates. This method is illustrated in FIG. 19.

The primary candidates are confirmed by an apoptotic assay and a ROSassay as described in Examples 2 and 3 respectively. Primary candidatescan be further tested to see whether they inhibit cell growth at G1 bymonitoring growth curve at a permissive temperature as in FIG. 1D and bymeasuring DNA content distribution using FACS analysis as in FIG. 2A. Toeliminate the possibility that a mutant cell is generated by thecandidate and thus forms colonies at the non-permissive temperature,cdc13-1 cells can be incubated with the candidate at about 37° C. forcolony formation. In one important aspect, the candidates will be testedfor whether they can improve mitochondrial function and inhibit cellloss in senescent mammalian cells such as WI38 fibroblasts. Whether theyare effective in preventing a particular age-related disease will thenbe further investigated in specific disease models. For example, theycan be tested for inhibiting tumorigenesis and/or decreasing braininfarction sizes in animal models.

Other types of suitable plates for the high-throughput screen include,but are not limited to, 384-well plates. In addition, this method can beadapted to other types of plates for detecting and confirming thecandidates, such as 6-well, 12-well plates and etc.

Example 2 Identification of Agents that Prevent cdc13-1 Cell Death Usingan Apoptotic Assay

Cell death in cdc13-1 exhibits apoptotic markers. This characteristiccan be employed for high-throughput screening since it takes less timethan the cell surviving assay mentioned in Example 1. It can also beused for confirming positives identified from the high-throughputscreening in Example 1. In addition, this assay can also be used toscreen for agents that inhibit apoptosis.

After incubating at a non-permissive temperature (about 37° C.) for aday (about 18-24 hrs), the cdc13-1 cells with compounds are stained withFITC-conjugated z-VAD-FMK (a suicide substrate that only binds toactivated caspase), or other suitable apoptotic detecting agents, for 20min in the dark. Cells may be washed once with Phosphate Buffered Saline(PBS). Plates are read by a fluorescent microplate reader. The negativecontrol (cells in the absence of the compounds or compositions screened)would have a high FITC signal due to cell death. The compounds orcompositions that reduce the FITC signals are considered as positives orprimary candidates. They are confirmed by assay the surviving cells asin Example 1, and further tested for their ability to reduce ROS andimprove mitochondrial function. These activities will also be tested inhuman telomere dysfunction models such as WI-38 senescent cells. Theiractivity in preventing cancer and other age-related diseases ordisorders are further tested in proper models.

The annexin V binding assay to measure phosphotidylserine flipping hasbeen described in FIG. 3B as an apoptotic marker. It can also be usedfor high-throughput screening.

Example 3 Identification of Agents that Prevent cdc13-1 Cell Death byMeasuring ROS (as Shown in FIG. 20)

Cell death in cdc13-1 exhibits dramatically elevated ROS levels. Thischaracteristic can be used to search molecules that inhibit cdc13-1 celldeath. The ROS assay takes less time than the cell surviving assaydescribed in Example 1, and can be used in the high-throughputscreening, as well as in confirming the primary candidates identifiedfrom high-throughput screening of Examples 1 and 2. In addition, thisassay can also be used to screen for antioxidants.

FIG. 20 illustrates the procedure. After incubating at a non-permissivetemperature (37° C.) for a day, the treated cells are stained withdehydrorhodamine 123 (or other suitable ROS detection agents) for aboutone hour in the dark. Plates are read by a fluorescent microplatereader. The negative control (i.e., cells in the absence of thecompounds or compositions screened) should have high levels of ROSreleased during cell death. The compounds or compositions that reducethe ROS are considered to be primary candidates, which are furthertested for cell death prevention and mitochondrial function improvementin both yeast and human models. Their activity in preventing cancer,neuron degeneration and other age-related diseases or disorders are thenfurther tested in proper models.

Example 4 Identification of Agents that Prevent Deterioration ofSenescent Cells in Telomere Dysfunction Models by MeasuringMitochondrial Mass

Cell death in cdc13-1 is accompanied with a dramatic increase inmitochondrial mass (“mito mass”). This characteristic can be used tosearch for molecules that inhibit cdc13-1 cell death. The mitochondrialmass assay takes less time than the cell surviving assay described inExample 1, and can be used in the high-throughput screening, as well asin confirming positives from high-throughput screen described inExamples 1, 2 and 3.

After incubating at a non-permissive temperature (about 37° C.) for aday, the treated cdc13-1 cells are stained with MitoTracker Green (orany suitable mitochondrial mass staining agents) for 20 min in the dark.Plates are read by a fluorescent microplate reader. Since dead cellscontain more deteriorated mitochondria which have lost membranepotential, they would show dramatic increases in mitochondria massstaining. The compounds or compositions that improve mitochondrialfunction and prevent cell death would reduce mitochondrialdeterioration, decrease mitochondrial mass signals, and thus areconsidered as positives. The positives will be further tested for theirability to prolong senescence in yeast and human models.

Example 5 Identification of Biological Molecules from DNA and NucleicAcid Libraries that Prevent Death of Senescent Cells

A Li-PEG transfection method is described as an example. However, othertransfection methods can also be used. Fresh yeast cells (cdc13-1) inlog phase are washed by distilled H₂O extensively. Cells are thenincubated with a transfection buffer (2 mM tris pH7.5, 100 mM LiAC, 0.5mM MgAC₂, 0.1 mM CaAC₂, 15% glycerol, 40% PEG-4000, 24 ug/mL ss DNA) anda DNA library at 24° C. for 1-4 hours. The mixtures are heat-shocked atabout 42° C. for 15 min. Three volumes of rich medium YEPD are thenadded to the mixture and incubated for an hour at a room temperature(about 24° C.). After centrifugation to remove liquid, cells arere-suspended in distilled H₂O and plated on the selecting medium platesfor the particular library. After incubation at the permissivetemperature 24° C. for at least 4 days, transformants are harvested andcultured in a liquid selecting medium at a permissive temperature (about24° C.). Cells in log phase are shifted to a non-permissive temperature(about 37° C.) and incubated for 2 days to allow cell death induced byinactivation of cdc13-1p. The cells are then properly diluted, plated onthe YEPD medium and incubated at about 24° C. for more than 4 days, oruntil surviving cells form colonies. The colonies are picked, and DNA isindividually isolated from each colony, amplified by PCR (DNA polymerasechain reaction) and sequenced to identify the DNA sequence. The DNA thatcan prevent cell death induced by inactivation of cdc13-1p is thenconfirmed by the introduction of the purified DNA into cdc13-1 cellsagain. The positives are then tested for prevention of deterioration ofsenescent human fibroblasts and regulation of mitochondrial function andoxidative stress.

Example 6 Identification of Proteins that can Promote Cell Death fromDisruption or Gene Deletion Libraries

To screen or identify proteins whose function is to promote telomeredysfunction-induced cell death, the gene disruption libraries, e.g.,yeast transposon insertion libraries can be introduced into cdc13-1strain. Alternatively, cdc13-1 can be introduced into yeast deletionstrain libraries, in which each strain has a specific gene deletion. Aprotein whose deletion or disruption can prevent cell death induced byinactivation of cdc13-1p is considered to be a candidate.

Example 7 Identification of Agents that Stimulate MitochondrialBiogenesis in Mammalian Cells

This experiment can be performed in human cells directly. Cell lines arechosen according to a particular age-related disease or disorderstudied. Cells in 96-well plates are treated for about a day and fixedby ethanol (final about 60%) to eliminate the effect of mitochondrialmembrane potential, and stained with a MitoTracker dye (Invitrogen). Thefluorescent signals are read in a fluorescent plate reader. Greater than20% increase in mitochondria fluorescent signal as compared tonon-treatment cells are considered as positives which are further testedfor their effects on preventing cell loss induced by telomeredysfunction in cdc13-1 and WI-38 models, and then on mitochondrialmembrane potential and ROS levels.

An alternative method to screen for candidates that stimulatemitochondrial biogenesis is usingreal-time-quantitative-reverse-transcription polymerase chain reaction(often donated as qRT-PCR) to test whether the mRNAs of transcriptionfactors for mitochondrial biogenesis are up-regulated. The transcriptionfactors for mitochondrial biogenesis comprise TFAM, NRF-1, NRF-2,PGC-1α, PGC-1β, TFB1M, TFB2M, ERRs (ERRα, ERRβ, ERRγ), PRC, POLRMT,PPAPs (PPAPα, PPAPγ, PPAPδ), and RIP140. Here TFAM is used as an exampleand primary human cells are used preferably. WI-38 cells seeded in96-well plates (or other plate formats) are incubated with a compoundlibrary for a desired period (e.g., about 18 hrs). Cells are washed withPBS and lysed in plates by the TaqMan Gene Expression Cells-to-CT Kitfrom Applied Biosystems, which removes DNA and makes cell lysates readyfor RT-PCR. Cell lysates are diluted in a new set of 96-well plates withreagents from the Qiagene's one-step qRT-PCR kits and QuantiFastMultiplex RT-PCR Kits. Primer sets ACAGCTAACTCCAAGTCAGATTATGTC-3′ and5′-GTAGACACTTGAGACTAACAACCGT-3′ for TFAM, and5′CAAAGACCTGTACGCCAACACAGT3′ and 5′-TTGCTGATCCACATCTGCTGGAAG-3′ forβ-Actin (control), that have been successfully used to detect TFAM mRNAincrease by 50 pM rapamycin and other agents (data not shown, and asdescribed in Fu X, et al, PLoS ONE, 3(4): e2009, 2008), can also beused. The compounds or compositions that increase TFAM mRNA greater thanabout 2-fold would be considers as positives, which are further testedfor senescence maintenance in the cdc13-1 or WI-38 telomere dysfunctionmodel.

Example 8 Testing of the Activity of a Compound or Composition inPreventing Senescence Loss in WI-38 Fibroblasts

There are situations in which a compound or composition such as a knowndrug, a nature extract or a nature product, a known peptide, or acandidate obtained from a library screening in a yeast model, needs tobe tested for whether it has an anti-aging effect in human cells. Thedesired testing can be done as described in FIG. 7 using WI-38fibroblasts.

Example 9 Determination of the Anti-Aging-Biological Concentrations ofRapamycin and its Derivatives Using cdc13-1 Yeast Cells

There are situations that the amount or concentration ofanti-aging-biologically active rapamycin needs to be determined, forexample, in different batches of purified rapamycin, samples during thepurification process, crude extracts, blood plasma after administrationof rapamycin, or in a chemically modified rapamycin derivative, etc. Todetermine the anti-aging-biologically active rapamycin from varioussources, the rapamycin containing materials are first diluted in aproper solvent with the desired serial dilutions (e.g., 3-fold or10-fold serial dilutions). About 5% (in volume, or less) of such dilutedmaterials are then added to freshly 10-fold diluted cdc13-1 cells inYEPD medium. After about 24 to 36 hrs incubation at the non-permissivetemperature (about 37° C.) to induce cell death, the number of survivingcells is measured by the colony formation assay as described in FIG. 1.To make a standard concentrations of active rapamycin, purifiedrapamycin is diluted with DMSO to 1 μM. Fresh cdc13-1 overnight cultureis 10-fold diluted in the YEPD medium. The 1 μM rapamycin is seriallydiluted 3-fold or 2-fold with the cell-containing medium to about 10 pM.The mixture is then incubated at about 37° C. for about 24-36 hrs forcell death induced by inactivated cdc13-1p. The numbers of survivingcells in the presence of various concentrations of standard rapamycinsolutions are determined by colony formation and the numbers are plottedagainst the concentrations to obtain a standard curve. The numbers ofsurviving cells in the presence of various rapamycin sources are thencompared to the standard curve, and the concentration of activerapamycin thus is determined. For a rapamycin derivative, the standardcurve would be first created for the same derivative. In addition tocolony formation, apoptotic assays and ROS release by cdc13-1 cell deathcan also be used to determine the anti-aging-biological concentrationsof rapamycin.

This method can also be used to determine the anti-aging-biologicalconcentrations of other anti-aging compounds or compositions, forexample, EGCG. In addition, this method can be used as an assay todetect the anti-aging activity of a biological sample.

Example 10 Detection of an Anti-Aging Agent Using the cdc17-1 andcdc17-2 Yeast Mutant

Rapamycin (1 and 3 nM) and reduced glucose are used as examples ofanti-aging agents. The cdc17-1 and cdc17-2 yeast cells was activatedfrom −80° C. stock first by streaking them on a fresh YEPD plate andthen incubating at a permissive temperature (about 24° C.) for 5 daysuntil single colonies formed. A few yeast colonies were picked andcultured overnight in the YEPD liquid medium at a permissive temperatureabout 24° C. The overnight cultures were diluted (10-fold dilution) intofresh YEPD medium containing rapamycin of 0, 1 and 3 nM, or 0.5% glucoseYEPD. The mixtures were then incubated at a non-permissive temperature(about 37° C.) for 22 hrs to induce cell death following the growtharrest triggered by to inactivate the DNA polymerase-alpha. The numberof surviving cells was measured by the colony formation assay. In brief,the mixtures were then serially diluted (10-fold), and a small amount ofcells (5 μl) was spotted on a YEPD plate. The plate was incubated at thepermissive temperature (about 24° C.) for at least 4 days to allowcolonies forming from surviving cells. As shown in FIG. 21A, rapamycinprevented cell induced by inactivation of DNA polymerase-alpha incdc17-1 and cdc17-2 mutant yeast.

Example 11 Detection of an Anti-Aging Agent Using the est1^(-ts) YeastMutant

Rapamycin is used as an example of anti-aging agents in this model.Rapamycin can prevent ROS induction and apoptotic-like cell deathinduced by inactivation of telomerase in the est1^(-ts) yeast cells(est1^(-ts) rad52::URA3) as described in my previous paper (Qi, H., etal., PlosOne, 2008). The assay was done by growing cells on therapamycin-containing plates. This assay can be modified by growing cellin the liquid medium to facilitate adoption of high throughput screen.In brief, est/^(-ts) cells are activated from the −80° C. stock first bystreaking them on a fresh YEPD plate and then incubating at a permissivetemperature (about 24° C.) for 5 days until single colonies formed. Afew yeast colonies are picked and cultured overnight in the YEPD liquidmedium at a permissive temperature about 24° C. The overnight culturesare diluted about 100- to 300-fold into fresh YEPD medium containing 0,or 1 nM of rapamycin. The mixtures are incubated at the non-permissivetemperature about 37° C. for about two days. Cells are diluted about100-300-fold again into fresh YEPD medium containing 1 nM rapamycin orthe control solvent and then incubated at the non-permissive temperatureabout 37° C. for about two days. The inactivation of telomerase at thenonpermissive temperature results in progressively shortening oftelomeres and eventually telomere dysfunction, which lead to ROSinduction and apoptosis-like cell death. Cell death can be measuredusing an apoptotic assay or a ROS assay. The ROS induction is measuredby incubating the cells with a dehydrorhodamine 123 solution (about 5μg/mL in a PBS buffer) in dark followed by FACS analysis. Cell death ismeasured by caspase activity as in Example 2.

B. Targeting the Nutrient/TOR/AMPK/Mitochondria/Senescence Pathway forPreventing and Treating Age-Related Diseases or Disorders

Example 12 Measuring the Components of the Senescence Pathway in aMammalian Model System

This example used Western blotting to determine the increases of keyproteins in the senescence pathway in mammalian cells induced by ananti-aging agent. Rapamycin was used as an example. WI-38 cells on the20th day after the last split (senescence) were treated with rapamycinof indicated doses for 18 hrs. Cell lysates were then analyzed byWestern blotting. The key proteins in the senescence pathway, p53, p21and pRB, were increase by the low doses of rapamycin (50 and 100 pM),but not by the higher dose 2000 pM (FIG. 9C).

Example 13 Agents that Exhibit Activities Against Age-Related Diseasesor in Cancer Chemoprevention Stimulate Mitochondrial Function andProlong Senescence

Experiments were done as described in FIGS. 7, 10 and 11. As shown inFIG. 10, 50 pM rapamycin, 250 μM AICAR, 20 μg/ml EGCG, 1.6 μg/ml GSE,reduced glucose (from 0.4% to 0.2%), 20 μg/ml bilberry extract (BE), 1μM AITC, and 12.5 μM 2-deoxyglucose increased mitochondria mass, anindicator of increased mitochondrial biogenesis, also prevented cellloss in senescent WI-38 fibroblasts (FIG. 7). Furthermore, phenethylisothiocyanate (PEITC), silibinin, selenite (Na₂SeO₃), and genisteinthat increased mitochondrial mass, also exhibited various degrees of theprotective effects on telomere-death in yeast cells (FIG. 10 and FIG.11).

Example 14 AMPK Activator Inhibits TPA-Induced Transformation of NIH3T3Cells

The experiment was done as in Example 19. In brief, 1500 NIH3T3 cellswere mixed with 50 μL of 0.4% agarose in culture medium (basal mediumwith 10% bovine fetal serum), layered on 96-well plates pre-covered with50 μL of 0.8% agarose in culture medium. 100 μL of culture mediumcontaining drugs to make final DMSO, 10 μM TPA, 40 μM AICAR, or 10 μMTPA+40 μM AICAR was then added into the wells. The cells were incubatedin a 37° C. incubator with 5% CO2 for more than 7 days. 50 μL of freshmedium was added every 5 days. Colonies were then counted under amicroscope. As shown in FIG. 12, TPA dramatically decreased themitochondrial mass in NIH 3T3 cells. The AMPK activator AICAR reversedthis decrease (FIG. 12). Furthermore, AICAR also reduced thetransformation of NIH 3T3 cells induced by TPA (FIG. 13A and FIG. 13B).

C. Use of a TOR Inhibitor as the Mimic of CR for Preventing and TreatingAge-Related Diseases or Disorders

TOR Inhibitors and Rapamycin. The specific TOR inhibitor rapamycin(Sirolimus) is the original member of a class of macrocyclic trienemolecules, comprising CCI779 (Temsirolimus), RAD-001 (Everolimus),AP-23573 (Deforolimus), AP-23675, AP-23841, ABT-578 (Zotarolimus),7-epi-rapamycin, 7-thiomethyl-rapamycin,7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin,7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-rapamycin,and 42-O-(2-hydroxy)ethyl rapamycin. Rapamycin was found originally tohave antifungal activity (Vezina, C., et al., J. Antibiot., 28:721(1975); Sehgal, S. N., et al., J. Antibiot., 28:727 (1975); Baker, H.A., et al., J. Antibiot., 31:539 (1978); U.S. Pat. Nos. 3,929,992, and3,993,749).

Rapamycin is used as an immunosuppressant (Santos, E. and Nebreda, A.R., FASEB, 3:2151-2163 (1989)): preventing or treating systemic lupuserythematosus (U.S. Pat. No. 5,078,999), pulmonary inflammation (U.S.Pat. No. 5,080,899), rejection in organ transplanting, arthritis(Carlson, et al., J. Pharmacol. Exp. Ther., 266:1125-1138 (1993);Foroncewicz et al, Transpl. Int., 18:366-368 (2005)), ocularinflammation (U.S. Pat. No. 5,387,589), and cardiac inflammatory disease(U.S. Pat. No. 5,496,832), preventing smooth muscle cell proliferationand intimal thickening following vascular injury (U.S. Pat. Nos.5,288,711 and 5,516,781) and is used on stent for preventing restenosis(U.S. Pat. No. 6,585,764). It is also patented for treating ocularconditions (U.S. Pat. No. 7,083,802), including age-related maculardegeneration (AMD) (U.S. Patent Application Publication Nos.20060182771, 20060247265, 20060263409, and 20070105761, 20060264453),choroidal neovascularization (CNV), and wet AMD (U.S. Patent ApplicationPublication No. 20050187241).

Rapamycin has been shown to have anti-proliferative and antitumoractivity. Rapamycin alone, or in combination with other drugs, has beenshown to have antitumor activity against adult T-cell leukemia/lymphoma(U.S. Pat. Nos. 4,885,171 and 4,401,653; European Patent Application525,960 A1), malignant carcinomas (U.S. Pat. No. 5,206,018), and anemia(U.S. Pat. No. 5,561,138). It can be used to treat metastatic breastcancer (U.S. Patent Application Publication No. 20070104721), neoplasms(U.S. Patent Application Publication Nos. 20040176339 and 20060035904),and early B cell derived acute lymphoblastic leukemia (U.S. Pat. No.7,026,330). It was also patented for treating tuberous sclerosis (U.S.Patent Application Publication No. 20050070567) and inhibiting abnormalcell growth in mammals (U.S. Patent Application Publication No.20060035907), reducing the proliferation and enhancing the apoptosis ofneoplastic cells (U.S. Patent Application Publication No. 20060094674),and treating proliferative and inflammatory disorders (U.S. PatentApplication Publication No. 20060135549), and chronic viral infection(U.S. Patent Application Publication No. 20070099844).

Use of rapamycin/mTOR inhibitors alone or in combination with otheragents has also been reported for treating various other diseases orconditions, such as diabetes mellitus (U.S. Pat. No. 5,321,009), skindisorders (U.S. Pat. No. 5,286,730), bowel disorders (U.S. Pat. No.5,286,731), neurological disorders, neurodegenerating diseases (U.S.Pat. No. 6,187,756), bone loss (U.S. Patent Application Publication No.20060173033), anti-angiogenic sustained release intraocular implants(U.S. Patent Application Publication No. 20070059336), and proteinconformational disorders by induction of autophagy (U.S. PatentApplication Publication No. 20070155771).

It was found to form complexes between TOR and FKBP12 and inhibit thecomplex formation between TOR and its normal substrate proteins such asthe TOR-raptor complex (TORC1). Inhibition of TORC1 formation results ininhibiting protein translation and ribosomal biogenesis, thus inhibitingcell cycle at G1 phase. TORC1 inhibition also leads to increasedautophagy for degradation of proteins and organelle for nutrients. Up todate, the reported uses of rapamycin at the therapeutic doses on variousdiseases are primarily based on TORC1 disruption and the subsequent cellcycle G1 inhibition and autophagy. In contrast, in the presentinvention, rapamycin and its analogs are used at low doses as a mimic ofcalorie restriction for the prevention or treatment of age-relateddiseases via the AMPK/Mitochondria/Senescence pathway.

Example 15 Low Doses of Rapamycin Inhibit TPA-Induced Transformation ofNIH3T3 Cells

Transformation of NIH 3T3 cells is a good in vitro tumorigenesis assay,which measures colony formation in soft agar (termed asanchorage-independent growth). The mutagen TPA(12-O-Tetradecanoylphorbol 13-acetate) is known to stimulate proteinkinase C (PKC), activate oncogenes and transform mouse embryonicfibroblast NIH3T3 cells. The TPA-induced anchorage-independent growth ofNIH3T3 cell was employed as a tumorigenesis assay. Interestingly, 10 μMTPA dramatically decreased the mitochondrial mass in NIH 3T3 cells, and1 nM rapamycin reversed this decrease as shown in FIG. 12. Furthermore,1 nM rapamycin also eliminated transformation of NIH 3T3 cells inducedby 10 μM TPA (FIG. 13). 1500 NIH3T3 cells were mixed with 50 μL of 0.4%agarose in culture medium (basal medium with 10% bovine fetal serum),layered on 96-well plates pre-covered with 50 μL of 0.8% agarose inculture medium. 100 μL of culture medium containing drugs to make finalDMSO, 10 μM TPA, 1 nM rapamycin, or 10 μM TPA+1 nM rapamycin was thenadded into the wells. The cells were incubated in a 37° C. incubatorwith 5% CO2 for more than 7 days. 50 μL of fresh medium was added every5 days. Colonies were then counted under a microscope. As shown in FIG.13A and FIG. 13B, 1 nM rapamycin totally blocked this process. As 1 nMrapamycin slightly slowed down the growth rate of NIH3T3, colonies werealso counted after 21-day's incubation to wait for the slowing growthcolonies. However, the same results were obtained. Therefore, the slowgrowth by 1 nM rapamycin is not the cause for elimination of colonyformation. It rather supports the role mitochondrial function inpreventing anchorage-independent growth or tumorigenesis via senescencemaintaining. In contrast, therapeutic doses of rapamycin have beenreported to promote tumorigenesis and thus increase the risk oflymphoma, skin cancer and other cancers in human. The antiproliferationactivity of rapamycin at therapeutic doses discovered recently is tolimit the growth of existing tumors, assumed by its inhibition in G1cell cycle and in protein synthesis

Furthermore, AICAR (40 μM) that activates AMPK and prolongs the lifespan of senescent primary fibroblasts of human (FIG. 7) also reversesthe decrease in mitochondrial mass triggered by TPA (FIG. 12), as wellas inhibits the anchorage-independent growth (FIG. 13A and FIG. 13B).These data further support the role of mitochondrial function inpreventing anchorage-independent growth or tumorigenesis. In conclusion,low doses of rapamycin and AICAR prevent tumorigenesis, at least in thecase induced by a mutagen TPA.

Example 16 Low Doses of Rapamycin Reduce ROS and Extend Life Span ofCultured GCN Neuron Cells

Cerebellar granule neuron (CGN) cultures were prepared from 7-day-oldrat pups. Briefly, the cerebellum was removed from the brain and placedin a Petri dish containing BMEM in 20 mM HEPES buffer (BMEM-HEPES).Cerebella were meninges and blood vessels were discarded to ensureminimal contamination from endothelial cells. Cerebella/cerebralcortices were then minced into fine pieces with dissecting knives andtrypsinized at 37° C. for 15 min. Trypsinization was inhibited by adding1 mL of BME containing 0.025% soybean trypsin inhibitor and 0.05% DNaseI. The tissue was gently triturated through a fire-polished Pasteurpipette until it was dispersed into a homogeneous suspension. Thesuspension was filtered through an ethanol-sterilized 40-μM mesh andpelleted by centrifugation. The pellets containing cerebellar granuleneurons were resuspended in B27 supplemented neurobasal mediumcontaining 25 mM KCl (Invitrogen, Carlsbad, Calif.). Cells were thenseeded into a 24-well plate (1 plate/cerebellum) and cultured inNeurobasal medium (Invitrogen) supplemented with B27, 20 mM KCl, 0.5 mMGlutamine, 100 units/mL penicillin, 100 μg/mL streptomycin. Rapamycinwas added to the culture 7 days. 31 days later, the MTT assay wasperformed to determine survival of neuron cells. As shown in FIG. 14A,the majority of CGN cells had already been lost after 31 days inculture. However, low doses of rapamycin prevented such a loss andextended the life span of the GCN cells in culture, but higher doses ofrapamycin did not have such as effect.

For ROS analysis, fresh isolated CGN cells in suspension culture inNeurobal complete medium were seeded in 12×75 mm tubes at a density ofone (1) million cells/mL/tube. Cells were treated with rapamycin for 20hrs and then stained with 2 μg/mL dehydrorhodamine 123 for 30 min priorto FACS analysis. As show FIG. 14B, rapamycin reduced the regular ROSlevel of CGN cells in suspension

Example 17 Low Doses of Rapamycin Reduce the Cerebellar Infarction Sizein a Rat Stroke Model

The middle cerebral artery (MCA) occlusion model of ischemic stroke wasused in this example. Spontaneously Hypertensive Stroke Prone (SHR-SP)rats were randomly divided into two groups (n=8 in each group): amatched control DMSO group and rapamycin group. They were anesthetizedwith 15% chloral hydrate (300 mg/kg, i.p.). Permanent focal cerebralischemia was induced by electrocoagulation of the distal portion of theMCA using a modified method described by Tamura and McGill (Tamura, A.,et al., J. Cerebral. Blood Flow Metab., 1:53-60 (1981). McGill, J. K.,et al., Stroke, 36:135-141 (2005)). Briefly, a segment of right MCAbetween the olfactory bundle and the inferior cerebral vein waselectro-coagulated. The coagulated artery was severed with microscissorsto ensure complete stop of blood supply.

Rapamycin and the control DMSO were administrated 10 minutes after MCAocclusion. Brain samples were harvested 24 h after MCA occlusion.Coronal sections of 2 mm in thickness were immediately stained with 2%2,3,5-triphenyltetrazolium chloride (TTC). The infarction region lookedpale, while the normal region looked red. The infarction area andhemisphere areas of each section (both sides) were traced and quantifiedby an image analysis system (Microsystems Type DM LB2, Leica, Germany).The possible interference of a brain edema in assessing the infarctionvolume was corrected for with a standard method of subtracting thevolume of the nonischemic ipsilateral hemisphere from the contralateralhemisphere volume. The infarction volume was expressed as a percentageof the contralateral hemisphere. The weights of the infarction tissueand the hemisphere were measured in a similar manner. As shown in FIG.15A, rapamycin at a low dose 10 μg/kg significantly reduced theinfarction volume induced by MCA occlusion. In contrast, rapamycin at anormal dose of 1 mg/kg did not have this effect, as reported in theliterature (Sharkey, J. J. and Butcher, S. P., Nature, 371:336-339(1994)). Furthermore, administration of rapamycin at doses of 0, 0.3, 1,3 and 10 μg/kg for 20 days prior to MCA occlusion also prevented thebrain damage (FIG. 15B).

Example 18 Low Doses of Rapamycin Reduce MPP+ Induced ROS

MPP⁺, an inhibitor of complex I (NADH CoQ1 reductase) in themitochondrial respiratory chain, is a dopaminergic neurotoxin. It iscommonly used to induce Parkinson's disease in marine models. Humanprimary fibroblast WI-38 cells were treated with 200 μM MPP for 3 days.Various concentrations of rapamycin were also incubated with cells for 3days as indicated in FIG. 14. Cells were then stained withdehydrorhodamine 123 in the dark for 30 min. Cells were then analyzed byFACS. Dehydrorhodamine 123 can be oxidized to rhodamine (that showsfluorescence) in proportion to ROS levels. As shown in FIG. 16, MPP⁺greatly increased ROS level. Low doses of rapamycin in picomolar (pM)ranges significantly reduced this increase.

Example 19 Low Doses of Rapamycin Reduce Myocardial Infarction Size in aRat Model

To determine Myocardial Infarction (MI) in Rats, Male Sprague-Dawley(SD) rats of 200 to 250 g by weight were used (n=10-12 for each group).Rapamycin at doses of 0, 10, or 100 μg/kg/day was administrated for 3days prior to the MI experiment. Under ether anesthesia conditions, theheart was exteriorized via a left thoractomy, and the left anteriordescending arteries were ligatured with 6-0 polypropylene suture betweenthe pulmonary outflow tract and left atrium. Then the beating heart wasquickly returned to its normal position, the thorax was closed, and theair was removed. Rats were returned to the cages with the previouslymentioned conditions. Five hours after the coronary artery ligature, therats were killed by an overdose of pentobarbital. The left ventricle wasisolated and cut into 4 to 5 slices perpendicular to the cardiac longaxis. The slices were stained for 30 min at 37° C. in a 0.1% solution ofnitro blue tetrazolium phosphate buffer and the MI size was measured asdescribed (Lin, L. L., et al, J. Cardiovasc. Pharmaco., 50:327-332(2007)). The normal tissue was stained in blue, while the necrotictissue remained unstained. The stained and unstained tissues wereisolated and weighed separately. The MI size was expressed as a fractionof the total left ventricular weight. As shown in FIG. 16, the low doseof rapamycin at 10 μg/kg/day significantly reduced the myocardialinfarction size, but not the higher dose 100 μg/kg/day.

Every patent and non-patent publication cited in the instant disclosureis incorporated into the disclosure by reference to the same effect asif every publication is individually incorporated by reference. Citationor identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

It is understood that the foregoing detailed descriptions andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention. Many variations of theinvention will become apparent to those skilled in the art upon reviewof this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.

What is claimed is:
 1. A method of preventing cell aging by extendingthe G0 phase in a cell in a subject in need thereof, the methodcomprising administering a low dose of a target-of-rapamycin (TOR)inhibitor to a subject, wherein: (a) said TOR inhibitor is rapamycin oran analog thereof selected from the group consisting of Deforolimus,AP-23675, AP-23841, Zotarolimus, CCI779/Temsirolimus,RAD-001/Everolimus, 7-epi-rapamycin, 7-thiomethyl-rapamycin,7-epi-trimethoxy-rapamycin, 2-desmethyl-rapamycin, and42-O-(2-hydroxy)ethyl-rapamycin, or a pharmaceutically acceptable saltthereof; and (b) said low dose of TOR inhibitor does not inhibit proteintranslation and cell growth at G1 phase of cell cycle.
 2. The method ofclaim 1, wherein said TOR inhibitor is rapamycin, or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 2, wherein the low doseof rapamycin is below about 1% of a therapeutic dose, wherein saidtherapeutic dose is in the range of 1 mg/day to 5mg/day.
 4. The methodof claim 3, wherein the low dose of rapamycin is below about 0.1% of thetherapeutic dose.
 5. The method of claim 3, wherein the low dose ofrapamycin is below about 0.01% of the therapeutic dose.
 6. The method ofclaim 3, wherein the low dose of rapamycin is below about 0.001% of thetherapeutic dose.
 7. The method of claim 1, wherein said subject is ahuman.
 8. The method of claim 7, wherein the low dose of rapamycin is inthe range from about 0.01 μg/day to about 50 μg/day.
 9. A method ofpreventing cell aging by extending the G0 phase in a cell in a subjectin need thereof, the method comprising administering a low dose of atarget-of-rapamycin (TOR) inhibitor in conjunction with administrationof a second agent to a subject, wherein: (a) said TOR inhibitor israpamycin or an analog thereof selected from the group consisting ofDeforolimus, AP-23675, AP-23841, Zotarolimus, CCI779/Temsirolimus,RAD-001/Everolimus, 7-epi-rapamycin, 7-thiomethyl-rapamycin,7-epi-trimethoxy-rapamycin, 2-desmethyl-rapamycin, and42-O-(2-hydroxy)ethyl-rapamycin, or a pharmaceutically acceptable saltthereof; (b) said second agent is independently selected fromantioxidants, antihypertensive agents, lipid-lowering agents,anti-stroke agents, anti-cancer agents, and different anti-aging agents;and (c) said low dose of TOR inhibitor does not inhibit proteintranslation and cell growth at G1 phase of cell cycle.
 10. The method ofclaim 9, wherein the TOR inhibitor is rapamycin, or a pharmaceuticallyacceptable salt thereof.
 11. The method of claim 9, wherein said secondagent is an antioxidant selected from vitamin C, vitamin E, betacarotene and other carotenoids, selenium, lipoic acid, lycopene, lutein,zeaxanthin, coenzyme Q10, glutathione, N-acetyl cysteine, melatonin,genistein, estrodiol, tea extract, and grape seed extract.
 12. Themethod of claim 9, wherein said second agent is another anti-aging agentselected from the group consisting of AICAR, EGCG, grape seed extract,bilberry extract, selenite, genistein, diallyl trisulfide, benzylisothiocyanate, phenyl isothiocyanate, phenethyl isothiocyanate,resveratrol, lycopene, and allyl isothiocyanate.
 13. The method of claim10, wherein the low dose of rapamycin is below about 1% of a therapeuticdose, wherein said therapeutic dose is in the range of 1 mg/day to5mg/day.
 14. The method of claim 13, wherein the low dose of rapamycinis below about 0.1% of the therapeutic dose.
 15. The method of claim 13,wherein the low dose of rapamycin is below about 0.01% of thetherapeutic dose.
 16. The method of claim 13, wherein the low dose ofrapamycin is below about 0.001% of the therapeutic dose.
 17. The methodof claim 9, wherein said subject is a human.
 18. The method of claim 17,wherein the low dose of rapamycin is in the range from about 0.01 μg/dayto about 50 μg/day.